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BCH2302

Biochemistry I

Laboratory Manual
Table of Contents
Introduction ..................................................................................................................... 1

Experiment 1: Solution Preparation in Biochemistry ....................................................... 2

Mini-Assignment 1: .................................................................................................... 10

Experiment 2: Titrations in Biochemistry ....................................................................... 12

Mini-Assignment 2: .................................................................................................... 27

Laboratory 3: Buffers in Biochemistry............................................................................ 30

Laboratory 4: Amino acids in biochemistry .................................................................... 39

In-Class Assignment .................................................................................................. 49

Laboratory 5: Bradford Assay ........................................................................................ 53

In-Class Assignment .................................................................................................. 61

Laboratory 6: Protein Extraction .................................................................................... 62

In-Class Assignment .................................................................................................. 67

Laboratory 7: SDS-PAGE.............................................................................................. 69

In-Class Assignment .................................................................................................. 75

Laboratory 8: Column Chromatography ........................................................................ 78

In-Class Assignment .................................................................................................. 86

Laboratory 9 Milk Chemistry (2 week lab) .................................................................. 88

In-Class Assignment .................................................................................................. 95


Introduction
Welcome to your Biochemistry I Laboratory course!

This course has been designed to take the skills you learned in Organic and General
Chemistry which focused on solutions, separations and basic chemical manipulations
and reactivity to give you a better understanding of the roles proteins and other
biomolecules play in biotechnology.

In order to understand the way biomolecules are purified, analyzed and manipulated,
we need to understand how they behave as chemicals, in short, we need to understand
the physical properties of the molecules to manipulate and take advantage of those
properties. We have designed this course to try to match the theory, although there are
aspects which will not match exactly.

The first four laboratories will focus on fundamentals of biochemistry acids, bases and
buffers. The importance of these aspects cannot be overstated. All living organisms
control the aqueous environment where their reactions take place. When manipulating
biochemical in the laboratory, we need to simulate, or manipulate, those conditions to
understand how processes are taking place.

Attention will then turn towards amino acids; the building blocks of proteins. Well
explore how they react in water, and how they can be used to determine the
concentration of proteins.

Once weve understood how these building blocks work, and their properties, well start
to manipulate them to isolate and purify items from the biological world. SDS-PAGE
(Sodium Docecylsulfate-Polyacrylamide Gel Electrophoresis) is a common method used
to separate and identify proteins, and forms the basis for the Western Blot. Once that
technique is covered, we will work on some food biochemistry by separating milk into
proteins, fats and sugars, and analyzing their content.

All of these techniques and ideas are crucial for any biochemistry and molecular biology
laboratory. Please try to ensure that you understand the lab: you get the most out of
these labs if you spend time understanding what you are doing, rather than going
through the motions.

See you in class!

1
Experiment 1: Solution Preparation in Biochemistry
Objectives:

1) Review concepts related to units of concentration and perform calculations


required for solution preparation
2) Demonstrate the ability to work with and convert between various units of
concentration.
3) Demonstrate the ability to perform solution preparation from solid reagents; and
from concentrated stock by dilution.

Introduction

Handling, preparing and performing calculations related to solution preparation are all
essential skills required in biochemistry. To gain employment in this field after
graduation, Technologists must be competent when preparing solutions (analytical
standards, buffers or any other solutions required in the laboratory).

For beginners, making solutions can be frustrating; this is an area in the lab where you
must use and hone your applied math skills.

The purpose of this lab is to review concepts covered in chemistry related to solution
preparation and ensure all students are comfortable with the required calculations, units
and nomenclature that you will encounter in this course, your studies and in your future
careers.

Solution Preparation:

Concentrations can be expressed in a wide range of units. A good laboratory


Technologist can navigate the various units that they may encounter in the lab, and
accurately prepare solutions when they are provided with a target concentration (in any
units) and a required volume.

In the field of Biochemistry, the most common concentration units you are likely to
encounter are:

1) Molarity (M): concentration that is expressed as moles of solute per liter of


solution. When preparing solutions in biochemistry it is common to use prefix
multipliers (Ex. m, , ) as solute concentrations are more commonly in the
millimolar (mM) or micromolar range (M). Where 1 mM = 1x10-3M, 1 M =
1x10-6M, 1 M = 1x10-9M.
2) Percent by weight (% w/w): Concentration that is expressed as the mass of the
solute as a percentage of the total solution mass.

Ex.

2
A 10 % by weight solution of glucose: has 10g of glucose, and 90 grams of
solvent for a total of 10 in 100 grams (or 10 %)

3) Percent by volume (% v/v): Concentration that is expressed as the volume of the


solute as a percentage of the total solution volume (Ex. Beer is approximately 5%
ethanol by volume)

Ex.

5% alcoholic beverage: contains 5 mL of concentrated ethanol per 100 mL


of solution

4) Weight per volume (w/v): Concentration is expressed in some unit of mass over
some unit of volume. Examples could include grams per liter (g/L) or mg/mL or
even ng/L

Ex.
5 % NaCl (w/v) solution: would have 5 grams of NaCl for every 100 mL
of total solution

Solutions prepared by dilution:

Stock solutions are a staple in the biochemistry laboratory. In order to save limited
space on the shelf, laboratory Technologists will prepare stock solutions with very high
solute concentration (buffers, dyes, nutrients for example). The concentrated stock
solution is stored until it is needed for a particular application; at that time, the laboratory
Technologist will take a portion of the concentrated stock, and use a small portion to
prepare a more dilute solution (by adding water) that will be used for application. We
call this a dilution from stock.

We see this same concept employed at the grocery store: In the freezer aisle you will
find concentrated frozen orange juice. The orange juice has been packaged and stored
as a highly concentrated stock in order to save valuable space in the store (some
laundry detergents are similar).

When a shopper buys the orange juice concentrate, they simply follow the instructions
on the label and add water to the stock concentrate in order to dilute the solution down
to a concentration that is acceptable for drinking. Orange juice is typically diluted from
the stock concentration in the can by a ratio of 1:4 (that is one part (or can) concentrate,
3 parts (or cans) water for a total of four parts). The juice has been diluted 1:4.

The working concentration of a solution is sometimes referred to as the 1X


concentration (i.e. the concentration of application drinkable orange juice).

3
Lets continue with our orange juice analogy; the canned concentrate is considered to
be a 4X solution (four times too concentrated); In order to achieve the appropriate
concentration for drinking, we must dilute to 1X or dilute the concentrate by a factor of
4.

It is not uncommon in the laboratory to prepare stocks that are as high as 50 or 100X
(read: 50 or 100 times) concentrated. When you need to use the reagent, you first must
dilute to the appropriate range.

In biochemistry, when we perform dilutions we generally use the following equation:

C1V1=C2V2

Where C represents the concentration (any units of concentration will work, Molarity is
common), and V is the volume (any units will work, units of Litre (L) are common). The
units of C1 and C2 must be consistent, and the units of V1 and V2 must be consistent.

When using this equation, there are a few things to remember:

Solution 1 will always be the more concentrated stock (C1>C2); always


Solution 2 will be the solution that you desire to make from the stock. Typically,
the volume (V2) and the concentration (C2) will be provided (but not always the
case).

At this end of this lab you will have performed the following:

Preparation of solutions
Dilutions of stock using the X notation
Dilutions by ratio (Ex. 1:2, 1:5, 1:10, 1:100)

Remember: solution preparation involves mastering a simple core set of skills that you
covered in level I and level II of the program

Ability to accurately weight reagents and make quantitative transfers


Ability to choose the best possible glassware for a desired solution preparation
o Must consider volume required and precision considerations
o Ex. Choosing a volumetric flask over a beaker!
Accurately measuring volumes of liquid with various glassware; or other
volumetric instruments (pipettes and micropipettes)
Performing calculations required to determine the quantity of stock solution and
the volume of diluent (typically water) that is needed to prepare a desired solution

4
Table 1: Laboratory Reagents

Reagent Handling PPE Disposal

NaCl For all reagents: For all reagents: For all reagents:

KCl - Wear appropriate - Lab coat, nitrile - DO NOT


PPE*. gloves and ASET dispose of down
approved safety the drain.
- Wash thoroughly glasses** must be
1 M acetic acid after handling. used for all reagents - Consult with
and protocols used in Professor for
- Handle in this lab. appropriate
fumehood only, disposal.
1M sodium acetate unless otherwise
indicated by your
Professor.
Na2HPO4 - Refer to
(Disodium MSDS/SDS.
phosphate)

KH2PO4
(Monopotassium
phosphate)

*PPE (personal protective equipment)

** Refer to ASETs Laboratory Safety Program

5
Protocol:

1. Complete the 3 parts of Worksheet #1 (below) with your partner before


proceeding to the lab activities.
a. Hand in the completed worksheet to your Professor before you leave the
lab
2. Prepare 100 mL of each of the following solutions, and show them to your
instructor:
a. 10 X Phosphate buffered saline buffer (PBS buffer). PBS contains 4
different solutes, all at different molarities. The solutes are NaCl, KCl,
Na2HPO4, and KH2PO4. All of these 4 solutes will go into the same
solvent (water) to make the final solution. The required concentration for
these 4 solutes are the following:
i. 1.4 M NaCl
ii. 27 mM KCl
iii. 43 mM Na2HPO4
iv. 14 mM KH2PO4
Molecular Weights: NaCl=58g/mol; KCl=75g/mol;
Na2HPO4=142g/mol; KH2PO4=136g/mol.
b. 0.1 M acetic acid (from 1 M stock)
c. 0.05 M sodium acetate (from 1 M stock)
d. Show all calculations
3. MINI ASSIGNMENT 1: Due at beginning of next weeks class

Worksheet #1:

6
Complete the entire worksheet prior to moving onto the active portion of the lab.
Confirm your calculations with the Professor. Your worksheet will be due at the
end of the lab period.

Table 1a: Molar Concentrations Complete table 1a. Calculate the molar mass
(g/mol) of each solute. Based on the concentration and the desired volume, calculate
the total mass that is required to make the solution. MWs: Na=23, Cl=35.5, O=16,
C=12, H=1

Concentr

solute in
Solution

Solution

Mass of

(grams)
Desired
Solvent

Volume

(#mols)
(g/mol)
Solute

Moles
ation
mW
(L)

1 Water 0.25 NaCl 0.1 mM

2 Water 1.0 NaOH 2.5 M

C6H12
3 Water 0.02 0.05 mM
O6

C2H5O
4 Water 25 30 mM
H

5 Water 1000 HCl 0.2 M

Show your work here:

Worksheet #1 (contd):

Table 1b: Mass to Volume ratio concentration Complete Table 1b. Calculate the
mass of the solute that is required based on the desired concentration (expressed in g/L

7
or other units) and volume provided. Notice the molar mass, and the total moles are not
needed for this type of concentration unit.

Solution Desired Mass of solute in


Solution Solvent Solute
Volume (L) Concentration (grams)

1 Water 50 NaCl 0.1 mg/L

2 Water 0.5 NaOH 2.5 g/L

3 Water 15 ALP 0.05 mg/mL

4 Water 0.005 C2H5OH 3 g/L

5 Water 1000 HCl 0.2 g/L

Show your work here:

Worksheet #1 (contd):

Table 1c: Concentration as a mass percentage Complete Table 1c. Calculate the
mass of the solute that is required based on the desired concentration (% w/w) and total
mass of solution provided. Notice the molar mass, and the total moles are not needed
for this type of concentration unit.

8
Total mass of Desired
Mass of solute in
Solution Solvent solution Solute Concentration
(grams)
(grams) (w/w %)

1 Water 5000 NaCl 5

2 Water 50 NaOH 10

3 Water 25 C6H12O6 1

4 Water 10 C2H5OH 3

5 Water 1 HCl 0.05

Show your work here:

9
Mini-Assignment 1:

Due at start of next weeks class: Make sure to show your calculations

Dilution calculations:

1) You are provided with a 10X solution of Tris buffer. From this stock you are
asked to prepare 100 mL of a 1X solution. What is the volume of stock solution
required, what is the volume of water required?

2) You are provided with a 12.5X solution of acetate buffer, you are asked to
prepare 50 mL of a 1X solution. What is the volume of stock solution required,
what is the volume of water required?

Dilutions when ratios are used:

1) You have a stock solution of NaCl that is 5M. You are asked to dilute the
solution 1:10 to a final volume of 150 mL. Calculate the concentration of the new
solution, calculate the volume of the stock and the volume of water that is
required.

2) You are provided with a 2.5M KCl solution. You are asked to dilute the sample
1:10, followed by a second 1:10 dilution and a final 1:10 dilution (serial dilution).
a. Sketch this dilution series
b. Calculate the concentration of the final sample

10
Appendix:

Some important review material related to solution preparation has been included in this
appendix.

From: Principles of Chemistry, Nivaldo Tro, 2006

11
Experiment 2: Titrations in Biochemistry
Objectives:

4) Review concepts related to chemical equilibrium, stoichiometry and acid-base


titrations
5) Complete a potentiometric titration (monoprotic and polyprotic acids)
6) Use a simple potentiometric titration to determine equivalence point, the half
equivalence point, and the pKa for weak acids (monoprotic and polyprotic)
7) Be able to construct titration curves and identify equivalence points and half
equivalence points

Introduction: Determination of pKa and Ka by titration

A potentiometric titration is a method in which the potential (voltage) between two


electrodes on a standard pH probe is measured (referent and indicator electrode) as a
function of the added reagent volume (titrant).

In a potentiometric titration of a weak acid with a base, one does not need to measure
the pH versus volume of the added titrant; instead the overall cell voltage is measured
and related to volume of titrant added (this can be achieved by changing the mode on
a standard pH probe).

The latter method is utilized to avoid errors to the pH caused by dilution effects, and is
generally considered to be more sensitive.

In this lab you will perform the titration of two weak acids: acetic acid (CH3COOH) and
phosphoric acid (H3PO4). The goal of this experiment will be to experimentally
determine the value of their pKa(s). You will be responsible for measuring the pH as a
function of titrant added, as well as the voltage as a function of the titrant added.

You will be asked to produce titration curves for monoprotic (weak acid with single
proton available for donation) and polyprotic weak acids (having multiple protons to
donate) and to perform some simple data manipulation in excel once the data is
collected.

12
Figure A: Titration curve for a generic acid (diprotic) with distinct pKa values

In Figure A, shown above, a plot of pH as a function of volume of titrant added is shown.


In this particular titration curve, we can observe two separate equivalence points.
Recall: the equivalence point (not to be confused with the end point (determined with
indicators) is the point in a chemical reaction between an acid and base where the
concentration of the acid is equal to the concentration of the base added
(stiochiometrically equivalent). Note: pH changes (or voltage changes) are most rapid
near the equivalence point.

For polyprotic acids (multiple protons to donate), there will be multiple equivalence
points. In this generic example, we observe two separate equivalence points that are
found by identifying the inflection point(s) on the titration curve. An inflection point is
the place on the curve where the curve changes concavity (in this case concave up to
concave down).

In addition to the equivalence point, we are also interested in the Half-Equivalence


Point. If the equivalence point indicates where the amount of added base is
stoichiometrically equivalent to the amount of weak acid present, then the half
equivalence point would indicate the point at which the weak acid and conjugate base
are in equal proportion. Do not confuse base added with conjugate base these are
2 different things!

At the half equivalence point [A-] = [HA]. Therefore, at this important point, the pH is
equal to the pKa (see Henderson Hasselbach Equation below)

[ A ]
pH pKa log
HA
The titration curve in Figure A is ideal. In practice, when the pH as a function of the
titrant added is monitored, the actual titration curve rarely looks this well defined (with
clear equivalence points and half equivalence points). This is the primary reason for
performing the potentiometric titration.

13
Recall:

The strength of an acid depends on the degree of dissociation when in solution.


For strong acids like HCl or H2SO4, the dissociation is virtually complete (Ka =
infinite), while in the case of weak acids, only a portion of the acid dissociates
(HA H+ + A-). In this case, equilibrium is reached.
o The value of the equilibrium constant Ka, defines the degree to which an
acid will dissociate (hence the strength):
HA + H2O H3O+ + A-

Ka
[H3O ] A
HA
Large values of Ka indicate more dissociation and thus greater acid
strength
pKa can also be used to predict acid strength:
pKa = -log(Ka)

In biochemistry, weak acids are often used to make buffers (we will see this more
next week). The useful range of the buffer depends primarily on the pKa of the
weak acid selected to make the buffer
o Generally, the range of application is between pKa 1

Buffering agent (weak acid) pKa useful pH range

Citric acid 3.13, 4.76, 6.40 2.1 - 7.4

Acetic acid 4.8 3.8 - 5.8

KH2PO4, 7.2 6.2 - 8.2

Notice how polyprotic citric acid has multiple pKa values. The pKa values can be
determined from performing titration experiments at a given temperature.

14
Table 1: Laboratory Reagents

Reagent Handling PPE Disposal

NaOH (solid) For all reagents: For all reagents: For all
reagents:

1.0 M acetic acid - Wear appropriate - Lab coat, nitrile


PPE*. gloves and ASET - DO NOT
approved safety dispose of
- Wash thoroughly glasses** must be down the drain.
0.1 M H3PO4 after handling. used for all reagents
and protocols used in - Consult with
- Handle in this lab. Professor for
fumehood only, appropriate
pH calibration unless otherwise disposal.
standards (4, 7, 10) indicated by your
Professor.

- Refer to
MSDS/SDS.

*PPE (personal protective equipment)

** Refer to ASETs Laboratory Safety Program - Refer to ASETs pH Meter Standard


Operating Procedure.

15
Protocol:

Calibration of a pH probe:

THIS EXPERIMENT WILL ONLY WORK IF YOUR pH PROBE is PROPERLY


CALIBRATED. See instructions attached at the end of the lab for calibrating the pH
probes.

Part I: Titration of a monoprotic acid (acetic acid - CH3COOH)

CH3CHOOH + H2O H3O+ + CH3COO- Ka = ?

Ka

[ H 3O ] CH 3COO
CH 3COOH
1. Obtain proper PPE including safety glasses.
2. Retrieve a pH meter, and pH calibration standards (4, 7, 10).
3. Calibrate the probe (see instruction at end of lab if needed).
4. In a flask or beaker obtain 200 mL of a 1.0 M NaOH solution (MW NaOH = 40
g/mol). This will be our titrant.
5. Obtain a retort stand with burette clamp.
6. Rinse a burette with a small amount of 1.0 M NaOH; drain the waste into the
base waste container, located in the fumehood.
7. Carefully: fill the burette with our 1.0M NaOH titrant solution.
8. Using the 1 M acetic acid sample provided by the instructor; prepare 100 mL of a
0.1 M acetic acid solution (by dilution) in a flask (use a serological pipette and a
graduated cylinder).
9. Accurately measure 20 mL of the 0.1 M acetic acid in a graduated cylinder and
transfer to a small beaker (100 mL for example).
10. Place the beaker containing acid on a magnetic stir plate and add a small stir
bar.
11. Carefully: lower the probe into the acetic acid solution BE SURE THAT THE
STIR BAR DOES NOT HIT THE TIP OF THE PROBE - THIS COULD BREAK
IT.
12. Record the initial pH in Table 1.
13. Change the mode on the pH probe and measure the voltage. Record the initial
voltage in Table 1.
14. Add 0.25 mL of titrant from the burette. It does not matter if you add exactly 0.25
mL, as long as you accurately record the volume in Table 1.
15. Measure and record the new pH and voltage in the Table 1. Repeat steps 14-15
until the pH changes become rapid. Once this happens, slow down the addition
of titrant to steps of roughly 0.1 mL.

16
16. Stop the titration when the addition of NaOH does not result in a significant
change in the pH.

Table 1: Titration of Acetic Acid:

Volume of NaOH (mL) added pH Voltage (mV)

0.0

17
18
19
20
Part II: Titration of a polyprotic acid (phosphoric acid H3PO4)

Weak polyprotic acids do not completely dissociate in solution (ie., do not lose all their
protons). The vast majority of the molecules remain unionized (H3PO4). Because
phosphoric acid is triprotic (has potential to lose up to three protons), those molecules
which do dissociate do so based on the series of equilibrium relationships shown below:

1) H3PO4 + H2O H3O+ + H2PO4-Ka1 = ?


2) H2PO4- + H2O H3O+ + HPO4-2 Ka2 = ?
3) HPO4 + H2O H3O + PO4
-2 + -3 Ka3 = ?

Where:-


[ H O ] H 2 PO4


[ H O ] HPO4
2

[ H O ] PO4
3

Ka1 3
H 3 PO4
Ka2 3

H 2 PO4

Ka3 3

HPO4
2

1. Prepare 100 mL of a 0.1 M NaOH solution (dilute the NaOH prepared in Part I
accordingly).
2. Empty and rinse the burette with a small amount of the 0.1 M NaOH.
3. Obtain 0.1 M phosphoric acid (provided) and transfer 20.0 mL to a 100 mL
beaker.
4. Carefully: fill the burette with our 0.1 M NaOH titrant solution (NOT 1.0 M!)
5. Place the beaker containing phosphoric acid on a magnetic stir plate and add a
small stir bar
6. Carefully: lower the probe into the phosphoric acid solution BE SURE THAT
THE STIR BAR DOES NOT HIT THE TIP OF THE PROBE - THIS COULD
BREAK IT.
7. Record the initial pH in Table 2.
8. Change the mode on the pH probe and measure the voltage. Record the voltage
in Table 2.
17. Using the burette, add 1.5 mL of the titrant to the beaker. As above, it does not
matter if you add exactly 1.5 mL, as long as you accurately record the volume (in
Table 2).
18. After the addition of each volume of titrant, record the pH and the voltage (in
Table 2). Repeat steps 17-18 until the pH does not significantly change upon
further addition of titrant. You may need to refill your burette a couple of times.
19. Notice: for Phosphoric acid, we expect to see two equivalence points (the
third is typically not observed).

21
Table 2: Titration of Phosphoric acid:

Vol. NaOH (mL) pH Voltage (mV)

0.0

22
23
24
25
26
Mini-Assignment 2:

Due at the start of class next week:

1) Plot a graph in excel for your acetic acid and phosphoric acid titrations showing
the pH (y-axis) as a function of titrant volume (x-axis). Label the acetic acid
titration as Figure 1a, and the phosphoric acid titration as Figure 1b. Print your
graphs. On your graph, estimate and label the:
- equivalence point(s). Draw a vertical line(s) down to the x-axis from this point(s).
- equivalence point(s). Draw a vertical line(s) down to the x-axis from this
point(s).
- pKa(s). Draw a horizontal line(s) to the y-axis from this point(s).

See the Pre-lab presentation for examples of what your curves should look like and how
they should be labelled. Below each graph, note the estimated value of the equivalence
point(s), equivalence point(s) and pKa(s). The equivalence and equivalence points
are in millilitres, and the pKa(s) are in pH units.

2) A more accurate method to depict the equivalence point is to use the voltage
readings. Using the voltage data you generated for both acetic acid and
phosphoric acid, plot the following information:
i) x-axis: volume of titrant added
ii) y-axis: change in mV over incremental volume of titrant added (mV/ V) as
shown in the example below (change is represented by the symbol) :

1.0 M NaOH (mL) pH Voltage (mV) V mV mV/V

0.00 3.00 237

0.50 4.24 163 0.50 74 148

0.70 4.38 155 0.20 8 40

0.90 4.60 142 0.20 13 65

etc etc etc etc etc etc


This is easily done in excel. This plot should clearly highlight the equivalence
points, which will appear on the graph as a peak(s). Print your graphs and label
the equivalence point(s). Draw a vertical line down to the x-axis from each
equivalence point. Using this line, estimate the volume of NaOH needed to reach

27
the equivalence point. Note the estimated equivalence point volume(s) below
each graph. Label your graphs as Figure 2a and 2b for acetic acid and
phosphoric acid respectively.(For more explanations on these graphs, see
Blackboard Course Content Pre Lab Presentations Lab 2: Titrations in
Biochemistry Pre-Lab Presentation.)

3) Compare the equivalence point derived from Figure 2a to Figure 1a (acetic acid).
How close are they? Which value do you trust more and why?
4) Compare the equivalence points derived from Figure 2b to Figure 1b (phosphoric
acid). How close are they? Which values do you trust more and why?

5) Using the estimated the pka(s) for acetic acid and phosphoric acid that your
determined using your graphs, calculate their respective Ka(s). Show your work.

Acetic Acid:

pKa: ________

Ka:

Phosphoric Acid:

pKa1:

Ka1:

28
pKa2: _______

Ka2:

Compare your data to the pKa reported in literature. Provide a short comment.

29
Laboratory 3: Buffers in Biochemistry
Objectives:

8) Review concepts related to the preparation and function of buffers


a. Buffer range
b. Buffer capacity
c. Types of buffers used in biochemistry
9) Evaluate and choose the appropriate buffers for biochemical applications
10) Perform calculations for buffer preparation

Introduction

The concept of using a buffer to resist pH change (or the change in the [H+] in solution)
is a topic that was covered in your chemistry courses in level I and level II of the
Biotechnology Advanced program.

In biochemistry, we think of the buffer as doing much more than simply maintaining the
solution pH. The buffers used in this field generally contain multiple solutes (generally
salts). These salts play a key role in buffering as well as providing co-factors for
enzymatic reactions, providing ionic strength (generally to provide isotonic conditions)
and in some cases providing cellular nutrients. Buffers are responsible for making the
aqueous phase hospitable for cells and their various products (proteins, nucleic acid,
etc). Because the majority of the applications in Biochemistry occur near to pH 7.0, we
generally prepare buffers that are most effective in this general range.

In biochemistry a wide range of buffers are used. The buffers you may encounter fall
under the broad headings:

1) Phosphate buffers Probably the most commonly used buffers in biochemistry.


Phosphate buffers generally have high buffering capacity (ability to resist pH
change), and are very useful in the range of 6.5 to 7.5. Because phosphate is
naturally found in cells its presence provides a more natural environment than
some other biochemical buffer options.

A common phosphate buffer is Phosphate Buffered Saline (or PBS). Although


various formulations exist, the buffer is made with various salts and phosphate
sources. A typical formulation is provided below:

The most common composition of PBS (1X)

Salt Concentration (mmol/L) Concentration (g/L)

NaCl 137 8.0

30
KCl 2.7 0.2

Na2HPO4 10 1.44

KH2PO4 1.8 0.24

2) Carboxylic acid buffers these buffers contain weak acids and conjugate base
pairs. Common carboxylic acid buffer systems are citrate buffer (citric
acid/trisodium citrate), acetate buffer (acetic acid/sodium acetate), etc. These
buffers tend to be most appropriate in the range of 3 to 6. Because carboxylic
acids are natural metabolites they may interfere with biological process in some
cases. These buffers are common in buffered fermentation applications where
pH is often below 7.0.

3) Zwitterionic buffers (A.K.A. Goods buffers) A zwitterion is a chemical


compound with acidic (proton donator) and basic groups (proton acceptor) in the
same molecule. Zwitterions are electrically neutral (no charge) but contain non-
adjacent regions of positive and negative charges. An example of a zwitterion
is an amino acid. Amino acids contain both a carboxylic acid functional group (-
COOH) and amine functional group (-NH2) (see the structure for a generic amino
acid below).

Figure A: Generic amino acid showing both a carboxylic acid and amine group. Amino
acids are one example of zwitterions.

In the 1960s zwitterions (and synthetic zwitterions) were recognized for their buffering
capabilities largely due to the work performed by N. E. Good. Good and colleagues
noted the disadvantages of conventional buffering systems, and outlined a series of
fundamental requirements of buffers for biochemical applications.

1) pKa between 6 to 8
2) Highly soluble in aqueous systems
3) Exclusion or only minimal transport by biological membranes

31
4) Minimal salt effects
5) Chemically stable
6) Insignificant light absorption in UV and visible range (230 nm 700 nm)

In his study, Good found that many synthetic zwiterionic species met these criteria
outlined above. In the table below a list of Zwitterionic buffers is included along with the
general application range.

Table A: Goods buffers and general application range

The most common zwitterionic buffer used in biochemistry is probably Tris. Choice of
buffer is generally application specific. The biggest downside to Goods buffers is
generally the cost.

32
NOTICE: Using the wrong buffer is a great way to ruin well designed experiments.

Did you know: Tris and glycine buffers (commonly used in biochemistry)
interfere with the Bradford Assay (a standard technique in protein
quantification (see future lab in BCH2301).

Both Tris and glycine are primary amines that interfere during the dye
binding process of the assay. This factor makes both Tris and Glycine
buffers unsuitable for protein based biochemistry in some cases.

This is an incredibly common mistake that even experienced lab workers


make because they dont understand the chemistry of the buffer.

Did you know: Some buffers are able to be autoclaved (withstand high
temperature and pressure during autoclave sterilization), while others
degrade with the application of heat.

This fact is easily overlooked. Get to know your buffers!

Choosing the correct buffer for an application depends on a number of considerations:

1) Technical application (are you doing protein work? DNA work? Other?)
2) Application pH (ideal range)
3) Ionic strength (osmolarity)
4) Temperature stability (can it withstand autoclave temperature and pressure?)
5) Chemical stability (storage considerations)

In this lab we will work with buffers from two of the categories outlined above. We will
test a phosphate buffer and a synthetic Zwitterionic buffer (Tris).

In the experiment we will explore preparation of common buffers. We will also explore
the buffer capacity and the temperature dependence of pH for various buffers.

33
Table 1: Laboratory Reagents

Reagent Handling PPE Disposal

NaCl For all For all reagents: For all


reagents: reagents:
KCl

0.1 M Tris buffer - Lab coat, nitrile


- Wear gloves and - DO NOT
0.5 M HCl appropriate Biotech/WWT dispose of
PPE*. Laboratory down the
0.5 M NaOH approved safety drain.
- Wash goggles** must
Na2HPO4 thoroughly after be used for all - Place waste
handling. reagents and into
NaH2PO4 appropriately
protocols used in
- Handle in this lab. labeled waste
KH2PO4 fumehood only, container,
unless located in the
otherwise fumehood.
indicated by NOTE: Change
your instructor. your gloves
immediately if
- Refer to they come into
MSDS. contact with any
of these reagents.

*PPE (personal protective equipment)

** Refer to ASETs Laboratory Safety Program

34
Protocol:

Part I: Prepare stock solutions

1) Obtain a pH probe.
2) Calibrate the probe.
3) Obtain 12 large test tubes. In each of the test tubes you will prepare a defined
mixture of sodium phosphate monobasic (NaH2PO4) and sodium phosphate
dibasic (Na2HPO4), as listed in Table 2 (below).

Table 2: Volumes of 0.1 M NaH2PO4 and 0.1 M Na2HPO4 to add to each of 12 test
tubes.

Sample # 0.1 M acid 0.1 M base pH measured predicted pH


(mL) (mL) (HH equation)*
(NaH2PO4) (Na2HPO4)

1 8.0 2.0

2 7.5 2.5

3 7.0 3.0

4 6.5 3.5

5 6.0 4.0

6 5.5 4.5

7 4.5 5.5

8 4 6.0

9 3.5 6.5

10 3 7.0

11 2.5 7.5

35
12 2.0 8.0

* Complete when preparing your Formal Lab Report See Formal Lab instructions
below

Part II: Buffer Capacity

The buffer capacity is defined as the amount of strong acid or amount of strong base
that must be added to 1.0 L of buffer to change the pH by 1.0 (lowering by 1.0 upon the
addition of strong acid or raising the pH by 1 upon the addition of a strong base).

1) Prepare 200 mL of 1X PBS buffer in an Erlenmeyer flask (notice the instructions


below are for 1.0 L of solution > show calculations). Use a magnetic stir bar to
thoroughly dissolve solutes.

The most common composition of PBS (1X)

Salt Concentration (mmol/L) Concentration (g/L)

NaCl 137 8.0

KCl 2.7 0.2

Na2HPO4 10 1.44

KH2PO4 1.8 0.24

2) Divide your 200 mL of PBS into two 100 mL parts; each in a large beaker (eg.,
200 mL size).
3) Drop a stir bar into one of your beakers of PBS, and place the beaker on a
magnetic stir plate and gently mix. Set the other aside for now.
4) Measure the pH of the buffer using a pH probe.
5) Fill a burette with 25 mL of 0.1 M HCl (you will have to dilute the stock
accordingly).
6) Note the exact starting volume of your HCl, and the starting pH of your PBS.
7) Slowly add the HCl from the burette to the beaker in 0.2 mL steps. Measure and
record the volume and pH after each small addition.
8) Note the total volume required to lower the pH of the buffer by 1.0

36
9) Calculate the number of moles of acid required to reach this pH = 1.0
10) Multiply the # moles by 10 (since we are using 100 mL instead of a litre).
11) Record the acid buffering capacity of the PBS buffer prepared.

______________________________________________________________________
_______

12) Place the second beaker with 100 mL of PBS on the stir plate and gently mix with
a magnetic stir bar.
13) Rinse your burette and fill with 25 mL of 0.05 M NaOH (you will have to dilute the
stock accordingly).
14) Measure the original pH of the buffer using the pH probe, and note the exact
starting volume of NaOH in your burette.
15) Slowly add the NaOH from the burette in steps of 0.2 mL, noting the change in
pH after each small addition. Be sure to also note the exact change in NaOH
volume with every step.
16) Note the exact volume required to raise the pH of the buffer by 1.0
17) Calculate the number of moles of base required to reach this pH = 1.0
18) Multiply the # moles by 10 (since we are using 100 mL instead of a litre).
19) Record the base buffering capacity of the PBS buffer prepared.

Part III: Impact of temperature on buffer pH

Buffers should always be prepared at the temperature at which they will be applied. In
this short study we will measure the pH as a function of changing temperature.
(Remember, means change in).

1) Obtain approximately 150 mL of the prepared Tris buffer.


2) Separate the 150 mL into three identical beakers.
3) Place the first beaker (50 mL) in a hot water bath at 50 oC for 20 minutes.
4) Place the second on the bench top (room temp).
5) Place the third in a beaker filled with ice for 20 minutes (replenish the ice as it
melts).
6) Measure the temperature and the pH of each of the buffers after 20 minutes has
elapsed.

Formal Lab Instructions:

The following are some preliminary instructions on what to include in your Results
section of your formal lab report. The instructor will be posting a more detailed version
shortly.

37
From Part 1:

1) Using your measured pH values, plot pH (y-axis) versus log(base/acid) as a


scatter plot in Excel. Notice the log values will range from positive to negative.
Using a line of best fit, determine the pH where the line of best fit intersects the y-
axis. This value is the pKa of the weak acid (measured experimentally).

2) Complete the predicted pH column of Table 2, using the Henderson-


Hasselbach equation:

bas e
pH pKa log
acid

Note: At 25oC the pKa of sodium phosphate monobasic is 6.82.

3) In the discussion of your formal lab, compare and contrast the predicted versus
measured pH values. What could be some reasons for differences between the
predicted pHs and the ones you measured experimentally?

From Part 2:

1) Using Excel, plot a graph of pH (y-axis) versus volume of titrant added (x-axis) for both
your HCl and NaOH titrations. Add a trend line for the data points. At the on the line
where the pH has changed exactly by 1.0 unit, draw a vertical line down to the x-axis.
Note the point at which the line intersects the x-axis, and use this information (ie.,
volume) to calculate the acid and base buffering capacity of your PBS.

From Part 3:

1) Using Excel, plot the pH (y-axis) as a function of the temperature (x-axis). Add a
trend line to the data points, which will hopefully reveal a trend depicting the
relationship between temperature and pH. Discuss this trend in the discussion
section of your report.

38
Laboratory 4: Amino acids in biochemistry
Objectives:

11) Explore concepts related to amino acids


a. structure
b. pKa
c. pI
12) Construct a titration curve
a. half equivalence points, pKas and isoelectric points
13) Use a titration curve to identify an unknown amino acid

Introduction
In all living organisms, there are four major classes of macromolecules;

Nucleic acids
Carbohydrates
Lipids
Proteins

Biological macromolecules can be thought of as massive molecules that are made up of


a series of smaller organic building blocks. In reality, Nucleic acids, Carbohydrates
and Proteins are biopolymers that are made by joining together smaller subunits
called monomers.

DNA molecules are a biopolymer made from smaller subunits called


nucleotides.
Carbohydrates or polysaccharides like cellulose or starch are made from simple
sugars like glucose.
Protein molecules are assembled by connecting a chain of amino acids. Each
pair of amino acids is connected by a peptide bond, and when a series of
peptides are linked we call the biopolymer a polypeptide (AKA. a protein).

In this lab we will focus on amino acids - the monomers that make up the most
interesting and diverse of all of the biological macromolecules: proteins.
Protein molecules are much smaller than DNA molecules but they are still large in their
own right. The size of proteins spans a range of about 10000 to 1 million daltons.
RECALL: A Dalton is equivalent to an atomic mass unit. So the molecular weight of a
protein can be as much as 1 million atomic mass units (keep in mind carbon is only
12)!!!

The diverse role of proteins:

Structure keratin (hair), collagen (connective tissues)


Transport myoglobin, hemoglobin (oxygen transport to muscle, organs)

39
Signalling hormones
Immune response, blood clotting
Catalysts biological catalysts (enzymes) are perhaps the most important
subgroup of proteins

All proteins are polymers made up of amino acid building blocks (monomers). The
generic structure of an amino acid is shown below:

Figure 1 - Generic amino acid showing both a carboxylic acid and amine group.

At near neutral pH, amino acids form zwitterions (overall electrically neutral species
where the amine group is protonated and the carboxylic acid group is deprotonated)

Figure 2: On the left, the amino acid is shown. On the right, the Zwitterion that is
formed at near neutral pH is shown

Zwitterions can accept or donate a proton (amino acids are amphoteric). The ionic form
of the amino acid will depend on the pH of the aqueous phase as shown below:
In acidic environment:

In basic environment:

In this experiment you will identify an unknown amino acid through the use of an acid
base titration. Included below is an example of a titration of the amino acid glycine.
Glycine has two dissociation steps that are evident on this titration curve. At low pH, the
H+ from the carboxylic acid is preferentially donated. The pKa associated with this
dissociation can be determined directly from the titration curve (as we did in previous
labs). The second dissociation occurs from the basic amino group at elevated pH.

40
The pKa for each of the dissociations can be determined from the titration curve by
determining the midpoint of each of the buffering regions (half-equivalence point). The
titration curve can also indicate where the amino acid behaves as a neutral salt. At this
point in the titration the amino acid is in the zwitterion form, possessing a net charge of
zero.
This point in the titration curve is called the isoelectric point (pI). Where pI can be
estimated as:
pI = * (pKa1 + pKa2)

Figure 3: Theoretical titration curve for the amino acid Alanine.


The titration curve can allow us to identify amino acids based on the following criteria:

Comparison of the pKa value and the pI values found experimentally with
literature values can allow for the identification of an amino acid source.

In total there are 20 common amino acids that when joined by a series of peptide bonds
produce a protein. A table of the 20 amino acids and common properties are shown
below:

41
Table 1: Amino acids and properties

In this lab you will first perform a titration of a known amino acid (glycine). You will
generate the titration curve in order to determine pKa 1, pKa2 and pI. The values
obtained will be compared with those found in literature.
Following the titration of glycine, you will be provided with one of three unknown amino
acids (aspartic acid, lysine or phenylalanine). You are encouraged to look up the
structure of each of the unknown amino acids prior to the lab. Some of the amino acids
are diprotic and some are triprotic (you can see seven of the amino acids in the table
are triprotic). This should be evident when you perform the titration on the sample.
RECALL (From Lab II):
Titration curves:

Figure 2: Titration curve for a generic acid (diprotic) with distinct pKa values

In Figure 1, shown above, a plot of pH as a function of volume of titrant added is shown.


In this particular titration curve, we can observe two separate equivalence points. The
equivalence point (not to be confused with the end point determined with
indicators) is the point in a chemical reaction between an acid and base where the

42
concentration of the acid is equal to the concentration of the base (stoichiometrically
equivalent).
Notice: pH changes (or voltage changes) are most rapid near this equivalence point.
In this curve there are multiple equivalence points. In this generic example, we observe
two separate equivalence points that are found by identifying the inflection point(s) on
the titration curve. An inflection point is the place on the curve where the curve changes
concavity (in this case concave up to concave down).
In addition to the equivalence point, we are also interested in the Half-Equivalence
Point. If the equivalence point indicates where the titrant added is stoichiometrically
equivalent to the amount of weak acid present, then the half equivalence point would
indicate the point at which the weak acid and conjugate base are in equal proportion.
At the half equivalence point [A-] = [HA]. Therefore, at this important point, the pH is
equal to the pKa (see Henderson Hasselbach Equation below):

43
Table 1: Laboratory Reagents
Reagent Handling PPE Disposal
0.1 M Glycine (pH adjusted with HCl; For all For all reagents: For all
pH=1.5) reagents: reagents:
- Lab coat, nitrile
0.1 M Unknown Amino Acid A (pH - Wear gloves and - DO NOT
adjusted with HCl; pH=1.5) appropriate Biotech/WWT dispose of
PPE*. Laboratory down the
0.1 M Unknown Amino Acid B (pH - Wash approved safety drain.
thoroughly after goggles** must - Place waste
adjusted with HCl; pH=1.5)
handling. be used for all into
- Handle in reagents and appropriately
0.2 M NaOH fumehood only, protocols used in labelled
unless this lab. waste
otherwise NOTE: Change container,
indicated by your gloves located in the
your instructor. immediately if fumehood.
- Refer to they come into
MSDS. contact with any
of these reagents.

*PPE personal protective equipment


** Refer to ASETs Laboratory Safety Program

44
Protocol:
Part I: Titration of glycine
Note: There is an in-class assignment that must be completed prior to leaving the
lab (see end of lab protocol). This assignment will make up part of your GLP
score show your instructor this assignment prior to leaving. (Graph paper
below).

1) Using pH standards, calibrate the pH probe


2) Prepare 100 mL of 0.2 M NaOH
3) Rinse and fill a burette with 0.2 M NaOH
4) Pipette 10 mL of glycine solution in a 100 mL beaker
5) Add 25 mL of distilled water
6) Place the beaker on a stir plate with a small magnet
7) Carefully: put the probe into solution
8) Measure and record the pH.
9) Titrate the amino acid solution with the NaOH solution in the burette by adding ~
0.5 mL of NaOH between readings. Note the exact amount added and the pH
each time. As you titrate, you are looking for the first equivalence point (where
the pH rises abruptly). As you approach the equivalence point, slow the rate of
titration down (add NaOH drop-wise) to accurately determine the titration curve.
RECORD ALL READINGS in the table in the worksheet.
10) After you pass the equivalence point, you can increase the rate of NaOH addition
(0.5 mL at a time)
11) Continue the titration until a final pH of about 12.5 is reached

Part II: Titration of unknown amino acid (you will perform this twice)

12) Refill burette with 0.2 M NaOH


13) Pipette 10 mL of unknown amino acid solution in a 100 mL beaker
14) Record the label of the unknown (A, B or C)
15) Add 25 mL of distilled water
16) Place the beaker on a stir plate with a small magnet
17) Carefully: put the pH probe into solution
18) Measure and record the pH
19) Titrate the amino acid solution with the NaOH solution in the burette by adding ~
0.5 mL of NaOH between readings. Note the exact amount added and the pH
each time. As you titrate you are looking for the first equivalence point (where
the pH rises abruptly). As you approach the equivalence point, slow the rate of
titration down (add NaOH drop-wise) to accurately determine the titration curve.
RECORD ALL READINGS in the table in the worksheet.
20) After you pass the end point, you can again increase the rate of NaOH addition
(0.5 mL at a time).
21) Continue the titration until a final pH of 12.5 is reached
22) Repeat the titration using the same unknown amino acid.
a. Use the first titration as a dry run; you should now know where to expect
the end points (when you repeat). Go slower as you approach the end
point to get the best possible titration curve.

45
Worksheet:
Part I: Glycine titration:
Volume of titrant (mL) (record cumulative pH
volume)
0

Part II: Unknown amino acid A:


Volume of titrant (mL) (record cumulative pH
volume)

46
0

Part II: Unknown amino acid B:


Volume of titrant (mL) (record cumulative pH
volume)
0

47
48
In-Class Assignment

Once you are finished both graphs, show your instructor

1) Plot the titration curve for glycine (pH vs. mL of NaOH added) on the graph paper
provided. From the curve determine pKa1, pKa2 and pI. Note the pKa1, pKa2 and
pI on your graph. (Recall: pI = [pKa1 + pKa2]/2)
2) For both unknown amino acids:
a. Plot the titration curves (pH vs. mL of NaOH added). From the curve
determine pKa1, pKa2 and pI. Note these values on your graph. Using the
table of amino acids in the introduction, guess the identity of the
unknowns.

49
50
51
52
Laboratory 5: Bradford Assay
Objectives:

1) Generate Bovine Serum Albumin (BSA) protein standards.


2) Use the Bradford assay to derive a standard curve using BSA standards.
3) Use the BSA standard curve to determine the concentration of 4 Unknown protein
samples.

Introduction:

Proteins have diverse functions ranging from lending structural support to cells,
catalyzing biochemical reactions and transporting molecules of various sizes. Scientists have
good reason to be interested in these properties of proteins. Some take advantage of the
catalytic nature of certain proteins by putting them to work in industrial bioreactors. Equally, in
biomedical research laboratories, scientists study how protein dysfunction can cause diseases.
In a clinical setting, scientists use some proteins as diagnostic markers, where their presence in
the blood indicates disease. Protein biochemistry is therefore often a key theme of
biotechnologists career, underscoring the importance of learning how to manipulate them in
the laboratory.

Being able to determine the concentration of protein in a given sample is arguably one
of the most important abilities of a protein biotechnologist. To illustrate this fact, lets consider
the example of a biotechnologist who works in a medical diagnostic laboratory. The
biotechnologist is tasked with screening the blood samples of 10 patients for the levels of
protein Z. Elevated levels of protein Z is considered a diagnostic feature of disease X.

The biotechnologist loads all the patient samples into the testing apparatus, and finds
that 3 individuals have elevated levels of protein Z. Does this necessarily mean they have
disease X? What if the samples from those 3 patients contained a higher amount of total
protein relative to the rest of the patients? This would give the illusion of elevated protein Z,
but when we account for total protein, levels of protein Z would be the same across all
patients (indicating no one has disease X). In order to determine with certainty if the 3
patients have disease X, the biotechnologist must first quantify the total protein
concentration of all patient samples.

There are numerous assays for quantifying the concentration of proteins in a sample,
and many rely on a chemical dye that changes colour upon interacting with proteins. In these
assays, a highly concentrated protein sample would undergo an intense colour change, whereas
a dilute sample would not dramatically change colour. This is well exemplified by the Bradford
assay, developed by Marion M. Bradford in 1976. In the Bradford assay, the Coomassie brilliant
blue r-250 dye (hereafter referred to as Coomassie dye) changes from red to blue upon binding
to proteins in solution. The article describing this method remains the 3 rd most highly cited
scientific publication of all time, highlighting its impact on the field of biochemistry.

53
Coomassie dye interacts with proteins by two different mechanisms. First, Coomassie
molecules are capable of degrading the tertiary structure of proteins by ionizing certain amino
acids that make up their peptide chains. This effectively degrades much of the proteins 3-
dimensional structure, allowing access to hydrophobic amino acid residues that are normally
buried on the inside proteins. Second, the Coomassie dye molecule possesses two sulfonic acid
functional groups that it uses to bond non-covalently to the basic R-groups of lysines, arginines,
and to some degree histidines. This changes the net charge of Coomassie to negative (anionic
state) resulting in a shift of its peak absorbance from 470 to 595 nm, where absorbance at 595
nm is roughly proportional to the amount of protein in a sample.

While the Bradford assay is extremely effective, it suffers some minor limitations. The
Coomassie dye does not bind to proteins of lesser size than 3000 Daltons, which can lead to
difficulty in quantifying preparations of small proteins (ie., peptides). Coomassie dye is also
incompatible with certain detergents commonly used in lysis buffers such as sodium
deoxycholate and sodium doedcyl sulfate (SDS). These compounds can interact with the
Coomassie dye in a way that produces a blue colour, which can lead to overestimating the
concentration of a protein sample. It is therefore important to use care when selecting a
method for protein quantification by observing the constituents of the medium in which the
protein sample is dissolved.

54
Materials:

TABLE 1: Required laboratory reagents. Proper handling, disposal and personal protective
equipment are to be used with all reagents.

Reagent Handling PPE Disposal

- Bradford reagent For all reagents: For all reagents: - DO NOT PUT DOWN
DRAIN.

- Bovine Serum Albumin - Wear appropriate - Lab coat, nitrile


(BSA) PPE*. gloves and - Bradford Reagent (mixed
Biotech/WWT with BSA protein) is to be
Laboratory approved disposed of in the
safety goggles** appropriately labeled
- Wash thoroughly must be used for all waste beaker for disposal
after handling. reagents and by Lab Technologist.
protocols used in
this lab.
- Refer to MSDS.

NOTE: Change your


gloves immediately if
they come into
contact with any of
these reagents.

*PPE = Personal Protective Equipment

** Refer to Laboratory Safety Manual

55
Protocol

PART 1: Setting up the Standards and Unknown protein samples.

1) Obtain eppendorf tubes, and label them 1 through 9.


2) Use Table 2 (below) to generate 5 standards and 4 Unknown protein samples.

TABLE 2: Reagent volumes required to generate 5 BSA standards and 4 Unknown protein
samples. All volumes are expressed in microliters.

3) Once you have completed preparing the Eppendorf tubes listed in Table 2, transfer the
contents of the tube to a clean cuvette. Add 2.5 mL of 1X Bradford reagent to each.
4) Carefully mix the contents of the tubes by agitating them gently.
5) Allow tubes to incubate for 5 minutes on bench top. While waiting, ensure the
spectrophotometer (Spec 20) is set to a wavelength of 595 nm.
6) Blank the spectrophotometer with Tube 1.
7) Read and note the absorbance of Tubes 2 through 9.

56
Part II: Determining the concentration of the BSA Standards and generating a Standard Curve.

1) In order to calculate the concentration of our Unknown protein samples, we must first
determine the concentration of our Standards. After all, we will use the Standards as a
reference for extrapolating the concentrations of the Unknown samples. Each Standard
is a dilution of the 2 mg/mL BSA stock (ie., 2 ug/uL or 2000 ug/mL) into a final volume of
500 uL, so simply use the C1V1 = C2V2 equation to complete Table 3 (below). You may
convert the units if you want its up to you! Be sure to accordingly label the axis of
Figure 1 and the estimated concentration column of Table 4 with the chosen units.

TABLE 3: Determining the concentration (C2) of the BSA Standards.

2) On Figure 1 (below), add a data point for each BSA Standard by plotting absorbance (at
595 nm) against concentration (C2). Add tick marks to further divide up the axes if
desired. Be sure to use as much of the space on the graph paper as you can.

57
3) Using a ruler (or something with a straight edge), draw a line of best fit through the data
points.
4) Draw dots on the line of best fit that represent the absorbances of each Unknown
sample (1 through 4).
5) From each dot, draw a vertical line down to the X-axis.
6) By observing where the vertical line intersects the X-axis, estimate the concentration of
Unknown samples 1-4. Add these estimated concentrations to Table 4 (below).

58
FIGURE 1. Bradford assay Standard curve generated by measuring 5 BSA standards at 595 nm.

59
Table 4: The concentrations of the Unknown protein samples 1-4, as estimated by extrapolation
off the BSA standard curve (Figure 1).

Part III: Estimating the true concentration of the Unknown protein samples.

1) Recall from Table 2 that we diluted all the Unknown protein samples in water before
assaying them. Therefore, our estimated concentrations (Table 4) actually represent a
dilution of our Unknown protein samples. To estimate their original concentration (C1),
use C1V1 = C2V2 to complete Table 5.

Table 5:

60
In-Class Assignment

(counts towards GLP):

1) Show your instructor the estimated true concentrations of your Unknown Samples
(Table 5). Also, show your instructor your standard curve graph, with the extrapolated
values for your unknown samples. Label the points on your curve.

2) Name 3 possible reasons that could explain any differences between your estimated
concentrations and the actual concentrations?

61
Laboratory 6: Protein Extraction
Objective:

1) Use different physical and chemical methods to lyse/extract protein from 3 distinct
biological matrices.

Introduction:

All known living organisms are made up of proteins, including all species of bacteria, fungi,
plants and animals. Even biological particles like viruses and prions are composed of proteins,
even though they are not considered truly living organisms! Many scientists are interested in
studying cellular proteins, which can be obtained from cells through a process called lysis. Cell
lysis occurs when the outer membrane of a cell ruptures, spilling its contents into the
surrounding medium. Released proteins can then be purified through centrifugation, and
subsequently used in experiments such as the Bradford assay, SDS-PAGE, Western blotting, and
ELISA (and others).

While the biochemical properties of proteins are generally similar across all organisms, the
outer cell membrane of bacteria, fungi, plant and animal cells is structurally very different.
Therefore, biotechnologists must choose appropriate physical/chemical cell lysis methods
based on the membrane rigidity of the biological material. Physical lysis methods include
blending, grinding, sonication or inducing rapid pressure changes to the medium. To
supplement physical methods, chemical detergents can be added to lysis buffers to enhance
cell rupture. Detergents are broadly classed as either ionic (eg., SDS, sodium deoxycholate) or
non-ionic (eg., NP-40, Triton X-100), where the type(s) selected will depend on the ensuing
downstream experiments. Ionic detergents are not suitable for protein interaction studies, as
these compounds interfere with protein-protein binding. However, non-ionic detergents are
often not sufficiently potent to solubilize membrane-integrated proteins, and are therefore
limited in this regard. Ideally, the chosen methods will be sufficiently strong to lyse most cells,
while not being excessively harsh so as to avoid damaging the released proteins.

Suspended animal cells (eg., blood cells or animal cell cultures) are by far the easiest material to
extract proteins from, as their cell membranes are delicate and susceptible to mild detergents.
More hardy methods must be employed when lysing intact animal tissues, as they are
supported by a specialized structure called the extracellular matrix. The extracellular matrix can
be thought of as a microscopic skeleton that runs throughout tissues to lend structural support.
Some tissues (such as brain) possess a delicate extracellular matrix, where passing tissue
through a needle in a mild detergent is sufficient for lysis. In contrast, to achieve the same for
muscle tissue, it needs to be frozen in liquid nitrogen and subsequently ground into a powder
with a mortar and pestle!

62
The relative ease of extracting protein from animal cells/tissues is contrasted by the robust
methods required to lyse bacterial cells. In addition to the plasma membrane(s), bacteria
possess a strong cell wall composed of polysaccharides and short amino acid polymers. For
protein extraction, biotechnologists commonly first treat bacteria with lysozyme - an enzyme
that weakens the cell wall by cleaving polysaccharide glycosidic bonds. Lysozyme treatment is
followed by harsh physical lysis methods such as sonication, which uses high frequency sound
waves to burst the bacteria.

Plant and yeast cells also possess cell walls, but their composition differs from bacteria. -
glucan polymers make up the majority of yeast cell walls, which can be broken down by
vortexing in the presence of small glass beads. The rigid cell wall of plant cells is composed of
cellulose, a massive polysaccharide consisting of several hundred glucose monomers.
Mechanical homogenization with a hand-held blender is an effective means to destroy plant
cell walls, allowing protein extraction from a variety of plant tissues.

In this laboratory session, you will lyse cells and extract protein from 3 biological matrices:
Escherichia coli, cultured mammalian cells and bovine liver. For E. coli, a combination of
lysozyme and sonication will be used. Cultured mammalian cells will be lysed by trituration in
an NP-40 buffer. Protein from bovine liver will be extracted by homogenization in NP-40 buffer.
These 3 protein samples will then be frozen until next weeks lab, where you will use them in
the Bradford assay, SDS-PAGE and Coomassie blue staining.

63
Materials:

TABLE 1: Required laboratory reagents. Proper handling, disposal and personal protective
equipment are to be used with all reagents.

Reagent Handling PPE Disposal

-E. coli culture, mammalian For all reagents: For all reagents: - DO NOT PUT DOWN
cells, bovine liver DRAIN.

- Wear appropriate - Lab coat, nitrile


- Lysozyme solution (20 PPE*. gloves and - Discard all lysis buffer
mg/mL egg white lysozyme Biotech/WWT waste in appropriately
in 1X PBS) Laboratory approved labeled waste beaker for
safety goggles** disposal by Lab
- Wash thoroughly must be used for all Technologist.
after handling. reagents and
- E. coli lysis buffer (50 protocols used in
mM Tris-HCl buffer [pH this lab.
8.0], 1 mM EDTA) - Discard all biological
- Refer to MSDS. material in biohazardous
waste receptacle.
NOTE: Change your
- NP-40 lysis buffer (50 gloves immediately if
mM Tris-HCl [pH 8.0], they come into
150 mM NaCl, 1% NP- contact with any of
40) these reagents.

- 1X PBS

*PPE = Personal Protective Equipment

** Refer to Laboratory Safety Manual

64
Protocol

PART 1: Protein extraction from an Escherichia coli culture. Maximum required time: 60 min.

The technologist has inoculated a liquid broth with E. coli, which has been incubating for
several hours at 37oC on a rotating platform. Once the culture reached an OD600 of
approximately 1.5, the bacteria were pelleted by high speed centrifugation, and resuspended in
a lysis buffer of 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA.

Specific materials/equipment required:

- Small beaker (~200mL)


- Ice bucket
- Aliquot of lysozyme solution
- Aliquot of E. coli suspension
- 2 microcentrifuge tubes
- P-1000 pipette and tips
1) Obtain 4 mL E. coli bacterial suspension from the instructor (in a 15 mL Falcon tube).
2) Add 400 uL of lysozyme (20 mg/mL) to the 4 mL of bacterial suspension.
3) Invert tube a few times, and incubate at 37oC for 10 minutes. When the incubation is
nearly finished, obtain a small beaker (~200 mL) filled with ice.
4) Place your tube in the center of the ice, remove the cap, and insert the sonicator probe.
If there are groups ahead of you, keep your sample on ice until it is your turn.
5) Ensure the sonicator probe is not touching the sides of the tube. Ensure the tip of the
probe is at least 1 mL below the surface of the bacterial suspension.
6) Put on safety head phones and start the sonicator. The instructor will have set the
sonicator to 3 x 10 second ON/OFF cycles (60 seconds total).
7) Once the sonication is complete, remove the tube from the apparatus and inspect the
bacterial suspension. It should be less turbid after sonication. Keep tube on ice.
8) Transfer 1 mL of sonicated bacterial suspension to a microcentrifuge tube.
9) Centrifuge at 15,000 x g for 10 minutes. Be sure to balance the centrifuge by opposing
your sample with that of another group.
10) While youre waiting, label a fresh microcentrifuge tube with (i) your names, (ii) your
laboratory section and (iii) E. coli protein. Place on ice.
11) Once the centrifugation is completed, transfer 500 uL of the supernatant to the fresh
microcentrifuge tube.
12) Freeze sample at -20oC for use next week.
13) Discard the previous tube in the biohazard waste receptacle.

65
PART 2: Protein extraction from cultured mammalian cells. Maximum required time: 45 min

Several days prior to this laboratory, the technologist seeded adherent mammalian cells into
tissue cultures dishes and incubated them at 37oC. On the day of the laboratory session, the
cells were enzymatically lifted from the dish by incubation with trypsin, and pelleted by
centrifugation. The cells were then resuspended in 1X PBS and kept on ice.

Specific materials/equipment required:

- NP-40 lysis buffer


- P-1000 pipette and tips
- P-200 pipette and tips
- Ice bucket
- 2 microcentrifuge tubes
- An aliquot of mammalian cell suspension
1) Obtain a _________ uL (ask instructor) aliquot of mammalian cell suspension in a
microcentrifuge tube. Label the tube with your initials.
2) Centrifuge the cells at 300 x g for 5 minutes.
3) Using a pipette, remove and discard as much of the supernatant as possible. Be careful
to not disrupt the cell pellet.
4) Add _________ uL (ask instructor) of NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 150
mM NaCl, 1% NP-40).
5) Set your P-200 pipette to a volume of 30 uL. Resuspend the cell pellet in the lysis buffer.
6) Vortex the suspension at full speed for 45 seconds. Incubate on ice for 5 minutes.
7) Centrifuge the tube at 15,000 x g for 5 minutes. Ideally, use another groups sample as a
centrifuge balance. Otherwise, oppose your tube with one with the same volume of
H2O.
8) Using a pipette, transfer as much of the supernatant to a fresh microcentrifuge tube.
Label the tube with (i) your names, (ii) your laboratory section and (iii) mammalian cell
protein.
9) Freeze sample at -20oC for use next week.

PART 3: Protein extraction from bovine liver. Maximum required time: 45 min

Specific materials/equipment required:

- 1 plastic weigh boat


- A glass homogenizer
- A small chunk of bovine liver
- Ice bucket
- A razor blade
- NP-40 lysis buffer
- 2 microcentrifuge tubes

66
- P-1000 pipette and tips
1) Obtain a small chunk (approximately 0.5 cm3) of bovine liver. As a sizing reference, this
chunk should be easily able to sit on your fingernail. Put it in the weigh boat.
2) Using a razor blade, carefully mince liver chunk into a slurry.
3) Put the base of the glass homogenizer in ice, and add 400 uL of cold NP-40 buffer.
4) Add the minced liver tissue to the homogenizer.
5) Using the plunger, grind the tissue in the lysis buffer until homogenized.
6) Transfer the lysate to a microcentrifuge tube labeled with your initials (on ice).
7) Load your liver sample into the centrifuge opposite to another teams sample (for
balance). If a group has taken an unusually large/small piece of liver, this could affect
the balance of the centrifuge. If there is any doubt about the centrifuge balance, check
with your instructor.
8) Centrifuge for 5 minutes at 15,000 x g.
9) Using a P-1000 pipette, transfer the supernatant to a fresh microcentrifuge tube. If you
cannot obtain the full 400 uL, collect as much as you can. There may be debris floating in
the supernatant, and if so, avoid transferring it to the fresh tube.
10) Label this new tube with (i) your names, (ii) your laboratory section and (iii) liver
protein.
11) Freeze sample at -20oC for use next week.

In-Class Assignment

Questions (Show instructor your answers prior to leaving lab GLP)

1) For each biological matrix, what were the physical and/or chemical methods used for
lysis?

2) Why should we expect the bacterial suspension to be less turbid after sonication?

3) Determine the concentration of lysozyme in your bacterial suspension during the


incubation at 37oC.

67
4) Name 2 alternative physical lysis methods for beef liver. Would these be more effective
than using the glass homogenizer?

5) Why is it important to keep protein lysates on ice during manipulations?

68
Laboratory 7: SDS-PAGE
Objectives:

1) Separate proteins by molecular weight using SDS-PAGE


2) Perform a Coomassie stain on an SDS-PAGE gel to visualize protein bands

Introduction:

Whole cell protein extract contains hundreds if not thousands of different proteins. To gain
insight into the individual constituent proteins of a given sample, scientists can employ
biochemical manipulations that separate proteins based on their molecular weight. Most
commonly, this is achieved with Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
(SDS-PAGE).

SDS-PAGE is founded on the principle that an electric current applied to uniformly charged,
linearized polypeptides will drive their migration through a porous matrix at a rate inversely
proportional to their molecular weight. Put simply, when all proteins of a sample are denatured
and carry the same charge, large proteins will migrate slowly through a gel whereas small
proteins will migrate rapidly.

This size-based rate of migration is dependent on two conditions. First, the 3-dimensional
structure of the polypeptides must be degraded so that they are linearized (like beads on a
string). Second, all proteins must be of uniform charge. This does not occur in the cell, since
proteins carry a charge related to their amino acid composition. If a polypeptide is composed of
mostly negatively charged amino acids (eg., aspartate and glutamate), its net charge will likely
be negative. Conversely, if the polypeptide is made up of many positively charged amino acids
(eg., arginine and lysine), the overall charge will most certainly be positive.

In order to render proteins linear and impart them with a uniform charge, they are heated in
the presence of SDS and -mercaptoethanol. -mercaptoethanol and SDS degrade protein
structure by reducing disulphide bonds of polypeptide chains, and interfering with charged-
based interactions that give polypeptides their 3-dimensional shape. Additionally, when SDS
binds to proteins, it coats them with a negative charge. The combined action of SDS and -
mercaptoethanol therefore results in the linearization of proteins and their adoption of a
negative charge proportional to their molecular weight (ie., a uniform negative charge, for our
purposes).

Now that our charge and shape conditions are satisfied, we can separate our proteins based on
their molecular weight using a polyacrylamide gel matrix. Polyacrylamide is ideal for resolving
proteins, as it is chemically inert, and therefore will not react with the proteins running through
it. As it polymerizes, it forms pores of a diameter that is inversely proportional to its acrylamide
percentage. High percentage gels will have small pore sizes whereas low percentage gels will
have larger pores. In this regard, large proteins will not travel efficiently through high

69
percentage gels, and as such, high percentage gels are typically used to separate low molecular
weight proteins. The opposite is true for low percentage gels.

To fuel the transit of polypeptides through a polyacrylamide gel, an electric current must be
applied to the SDS-PAGE apparatus. Recall that SDS treatment confers a uniform negative
charge to the polypeptides. Therefore, in an electric field, they will all migrate towards the
cathode (positive electrode). Typically, current must be applied to the apparatus for a minimum
of 45 minutes to result in appreciable protein migration through the polyacrylamide gel.

Once SDS-PAGE is complete, it is desirable to visualize the proteins to estimate their abundance
and/or relative concentration in a sample. This can be achieved by staining the gels with
Coomassie stain. Recall from Lab 5, that Coomassie dye is used in the Bradford assay when
quantifying protein concentration. Coomassie interacts with certain amino acids via a
mechanism that renders it blue in colour. This phenomenon can be applied to proteins
embedded in polyacrylamide gels, where Coomassie dye will stain the protein bands blue for
easy viewing. Background staining is removed with ample washing in water or destaining
solution.

In last weeks laboratory, we isolated total cell protein from E. coli, cultured mammalian cells
and bovine liver. This week, you will run your protein samples on an SDS-PAGE gel, which will
separate the constituent proteins by molecular weight. Then, we will stain the gels using the
Coomassie method, which will allow us to visualize the proteins embedded in the gel.

70
Materials:

TABLE 1: Required laboratory reagents. Proper handling, disposal and personal protective
equipment are to be used with all reagents.

Reagent Handling PPE Disposal

- 1X Running buffer For all reagents: For all reagents: - DO NOT PUT DOWN
(glycine, Tris base, sodium DRAIN.
dodecyl sulfate [SDS],
dH2O)
- Wear appropriate - Lab coat, nitrile
- SDS-PAGE gel (Tris-base, PPE*. gloves and - Discard all
SDS, ammonium Biotech/WWT solvent/buffers waste in
persulphate, dH2O, Laboratory approved appropriately labeled
Tetramethlethylenediamine safety goggles** waste beaker for disposal
[TEMED]) - Wash thoroughly must be used for all by Lab Technologist.
after handling. reagents and
- Laemmli buffer (Tris base, protocols used in
SDS, glycerol, bromophenol this lab.
blue, dH2O) - Discard all biological
- Refer to MSDS. material in biohazardous
- Coomassie stain waste receptacle.
(Coomassie brilliant blue G- NOTE: Change your
250, HCl, dH2O) gloves immediately
if they come into
- Protein samples from Lab contact with any of
6 (Tris base, NP-40, EDTA, these reagents.
H2O, NaCl).

*PPE = Personal Protective Equipment

** Refer to Laboratory Safety Manual

71
Protocol

PART 1: Loading and running the SDS-PAGE gel. Maximum time required: 80 mins

The technologist has set up SDS-PAGE gels in the gel running apparatus prior to the laboratory
session. Each apparatus holds 2 gels, and each gel will accommodate 2 teams (See Figure 1). So
you will be running your gel in the same apparatus as 3 other teams. Find other teams for this
purpose once youve reached Step 6 (below).

Specific materials/equipment required:

- Protein samples from last week


- Laemmli buffer (protein loading dye)
- Molecular weight ladder
- SDS-PAGE gel
- Ice bucket
- 6 microfuge tubes

1) Place your protein samples from last week on your bench-top to thaw.
2) As they begin to thaw, inspect the SDS-PAGE gel apparatus. Familiarize yourself with
where the gel is located, and plan a strategy for how you will load your samples into it
using a P200 pipette (Refer to Figure 1).
3) Return to your bench and accelerate the thawing of your protein samples by warming
them with your hands. Once they are completely thawed, keep them on ice.
4) Obtain 6 microfuge tubes and label them according to Table 2 (below). Add your initials
to each tube as well.
5) Prepare your protein samples in the microfuge tubes according to the reagent volumes
indicated in Table 2.

72
TABLE 2: Volumes required to prepare protein samples for SDS-PAGE. All volumes are
expressed in microliters.

6) Close tubes and place on a 95oC heat block for 5 minutes. In the meantime, find 3 teams
that you will run your samples with. (You will be running your gels in the same
apparatus).
7) Load your samples into the centrifuge, ensuring they are properly balanced. Perform a
quick spin of 1 minute at 5000 g.
8) Remove samples and bring them over to the SDS-PAGE apparatus.
9) Choose and note which gel is yours in the SDS-PAGE apparatus. This can be easily
confused if the apparatus is moved/rotated. One strategy is to place a piece of labeled
tape on the outside of the apparatus indicating which gel is yours.
10) Ask the instructor to add 10 uL of molecular weight ladder to the left-most well of the
gel, as depicted in Figure 1 (below).
11) Using a P-20 pipette, sequentially add 20 uL of your protein samples into the wells,
starting from the well adjacent to the ladder (as depicted in Figure 1).
12) Allow another team to load their samples beside yours (leave an empty well between
groups; see Figure 1).

73
13) Attach the lid to the apparatus. Run the gels at 150 V for 45 mins. If you are unsure of
your setup, ask the instructor.

FIGURE 1: Loading of SDS-PAGE gel with 25 uL of each protein sample from Table 2.

PART 2: Coomassie staining of SDS-PAGE gel from Part 1. Maximum required time: 50 min

Specific materials/equipment required:

- A piece of Saran wrap


- A blank piece of paper
- A small plastic box
- Coomassie stain
- Ample dH2O
- Your completed SDS-PAGE gel

1) Turn off the power source of the SDS-PAGE apparatus.


2) Disassemble the cassette, and remove the glass plates sandwiching your gel.
3) Separate the glass plates using a small metal spatula.
4) While being careful to not tear the gel, transfer it to a plastic box.
5) Use the metal spatula to cut off the stacking portion of the gel. Discard in garbage.
6) Cover the gel with dH2O (in box).
7) Place box (containing resolving gel) in microwave and heat for 30 seconds.
8) Place box on orbital shaker for 5 minutes.
9) Discard dH2O down the drain.
10) Repeat steps 6-9 two more times.
11) Cover gel with Coomassie stain and heat in microwave for 10 seconds.
12) Incubate gel on orbital shaker for 15 minutes.
13) Pour staining solution into the used stain bottle.
14) Cover the gel with dH2O, incubate on orbital shaker for 5 minutes.

74
15) Place your piece of blank paper on a flat surface, and place a piece of Saran wrap on top
of it.
16) Gently transfer your gel from the box to the Saran wrap.
17) Discard dH2O down drain, and observe the gel for protein bands.
18) Show your gel to the instructor.

In-Class Assignment

Questions for GLP

1) Do all lanes of your gel possess the same amount of protein? How can you tell?

2) How could the Bradford assay be combined with SDS-PAGE to improve the accuracy of
results?

3) Why are some bands on your SDS-PAGE gel faint whereas others are more prominent?

75
4) What would happen if we did not treat the samples with SDS prior to running them in a
polyacrylamide gel?

5) What is the purpose of the stacking gel?

6) What is the approximate molecular weight of the most abundantly expressed protein in
each of your samples?

7) Identify the most weakly expressed protein in each of your samples. What is its
molecular weight?

76
GEL VIEWING PAPER

77
Laboratory 8: Column Chromatography
Objectives:
1) Use two methods of column chromatography to separate biomolecules from
complex mixtures
a. Size based separations (gel filtration)
b. Charge based separations (ion exchange)
2) Perform simple assays to analyze protein fractions to verify the efficacy of each
separation scheme

Introduction:
In order to study a particular protein in the research lab, or produce a highly purified
protein in industry for monetary gain, biotechnologists must be able to target a specific
protein of interest and separate that protein from a complex mixture of biomolecules,
cellular components and other chemicals.
The key to performing separations in any field is to understand the properties of
the target component (in this case a protein of interest) as well as the properties of the
other components of a mixture in which the target is found. In order to perform the
separation successfully, it is important to exploit the property difference between the
target and the other molecules in the mixture. The larger the difference in
properties, the easier the separation will be.
A typical purification scheme may consist of many steps performed in a particular
sequence with the ultimate goal of obtaining the most pure fraction possible.
Some common techniques used in the lab for purifying a particular protein:
Protein chromatography (affinity separation)
Electrophoresis (size)
Gel filtration (size based separation)
Ion exchange chromatography (charge based separation based on isoelectric
point (pI) of the protein
Centrifugation (separation based on density)
Dialysis tubing (size)
Ammonium sulfate precipitation (solubility)

Remember: The steps required for separation and purification will vary from protein
to protein. When devising a scheme to purify a target protein we must consider the
biological matrices and the source of the protein, as well as the location of that
particular protein (intracellular, extracellular, etc).
Example: A particular protein may be purified by the following steps:
Crude cell extract > Centrifugation > Ammonium Sulphate Precipitation > Gel filtration >
Ion exchange chromatography
The goal is that with each step, the degree of purity of the fraction collected
increases.
A separation scheme like this would not be applicable in all cases. Often this means
that a unique protocol for purification has to be developed for each protein that we wish
to isolate. The design is often optimized over weeks or months.

78
In this lab the aim is to test two fundamental techniques used in purification of
proteins. In this lab we will separate a mixture prepared with three specific molecules.
Each lab group will be provided with a sample mixture that consists of:
1) Catalase (1 mg/mL)
2) Cytochrome c (1 mg/mL)
3) Riboflavin (0.01 mg/mL)

In part I, students will pack a column and perform size exclusion chromatography.
Gel filtration is used to separate proteins of different sizes. The separation consists of
packing a column with a stationary phase. A mixture of proteins or molecules are
introduced to the stationary phase and a liquid (or mobile phase) is used to drive the
mixture through the column.
The stationary phase consists of porous beads that act as a packing. The mobile
phase is typically a buffer. A sample mixture containing proteins is introduced on the
top of the column is allowed to pass through the stationary phase either by gravity or by
providing pressure (manually or with a powerful pump).
Small molecules have the ability to occupy the pores of the packing (stationary phase).
When they occupy the pores, this causes these small molecules to travel slowly as they
traverse the column packing. Larger molecules do not enter the pores to the same
degree and therefore travel at a faster rate through the column.
Molecules are therefore eluted in order of decreasing molecular size.
In part II, students will pack a column with an ion exchange resin and perform ion
exchange chromatography separation.
Ion exchange chromatography uses charged stationary packing to bind or
capture oppositely charged proteins in a mixture as they pass over the packing. A
sample mixture containing proteins is introduced on the top of the column is allowed to
pass through the charged stationary phase either by gravity or by providing pressure
(manually or with a powerful pump).
The charge of each of the proteins, and thus their interaction with the packing will
depend on the pH of the solution and the properties of the particular protein (pI).
Ion exchange equilibrium consists of four basic steps:
Ion exchange chromatography is defined as a reversible adsorption of charged
molecules (in this case: proteins) to immobilized ion groups on a matrix (stationary
phase) of opposite charge. Separation can be achieved by first adsorbing target
proteins and later releasing these proteins by manipulating the ionic strength of the
elution buffer.
1) Equilibration of the ion exchange resin (stationary phase):
a. This step involves preparing the resin in a buffer that has an application-
specific pH and ionic strength.
2) Introduction of sample:
a. Molecules with the correct charge will bind to the stationary phase
b. Uncharged molecules travel through rapidly and are eluted with the buffer
3) Molecules are eluted using a gradient concentration of NaCl:
a. NaCl concentration is increased in the buffer and this causes weakly
bound proteins to elute first.
b. As the concentration of salt increase the more strongly bound proteins will
elute.

79
4) A high concentration salt solution is used to elute the most tightly bound
macromolecules. The high concentration salt solution will also regenerate the
column packing for reuse.

For the separation in this lab, we will be using Sepharose cation exchange resin that
has a negative charge around neutral pH. The separation will depend upon the
difference of charge of the proteins that are to be separated.
Recall: The pI can be used to predict the charge of a protein. At a pH below the pI, the
protein will carry a net positive charge. At a pH above the pI of the protein, the protein
will carry a net negative charge.
Elution of the bound protein can occur by increasing the ionic strength of the
buffer. In the case of Sepharose, NaCl, which contributes the counter ion (Na+), will
cause competition for the adsorption sites and dislodge the positively bound proteins as
the concentration of ions is increased.
Elution is typically done with a gradient concentration of ions (increasing from low
ionic strength to high ionic strength), or by using step-wise elution (ex. sequentially
using three different buffers with increasing ionic strength). In this lab we will use a
step-wise elution scheme.

Some properties of the proteins relevant to our current experiment are summarized
below:
Molecule Molecular Weight Isoelectric Point (pI) Details
(Daltons)
240000 Negatively charged at Enzyme that produces
Catalase pH 7.5 water and oxygen from
hydrogen peroxide.
Colourless.
Cytochrome c 12000 Positively charged at pH Transports electrons
7.5 across cell membranes.
Strong red colour
Riboflavin 376 Neutral at pH 7.5 Also called vitamin B12.
Yellowish in colour

80
Table 1: Laboratory Reagents
Reagent Handling PPE Disposal
Size exclusion resin For all reagents: For all
(Sephadex G25 or For all reagents:
Sephacryl) reagents: - Lab coat, nitrile
Ion exchange resin gloves and - DO NOT
(Sepharose) - Wear Biotech/WWT dispose of
Mixture: appropriate Laboratory down the
o Riboflavin (0.01 PPE*. approved safety drain.
mg/mL) - Wash goggles** must - Place waste
o Cytochrome c (1 thoroughly be used for all into
mg/mL) after handling. reagents and appropriately
o Catalase (1 mg/mL) - Handle in protocols used in labelled
50 mM Tris-HCl fumehood this lab. waste
buffer(pH=7.5) only, unless NOTE: Change container,
Medium Salt Buffer (50 mM otherwise your gloves located in the
Tris-HCl [pH=7.5] with 50mM indicated by immediately if fumehood.
NaCl) your they come into
instructor. contact with any
High Salt Buffer (50 mM Tris
- Refer to of these
HCl [pH=7.5] with 500mM
MSDS. reagents.
NaCl)
10% hydrogen peroxide SAFETY: the
hydrogen
peroxide is an
oxidizing agent.
Handle and
dispose with
care!
*PPE (personal protective equipment)
** Refer to Laboratory Safety Manual

81
Protocol:
NOTES: READ AND UNDERSTAND THESE NOTES BEFORE STARTING
1. The resin in your column should never go dry at any point as this will ruin it. To
prevent this from happening, always ensure a small volume of buffer is present
just above the gel surface. To prevent the column from going dry, you can cap
the bottom of the column at any time.
2. GENERAL COLUMN MANIPULATION: To be followed once you have added
sufficient gel slurry to your column.
a. Ensure there is a waste beaker underneath your column. Remove the top cap.
b. Attach a length of flex tubing to the top cap.
c. Obtain a large syringe and pull the plunger out to its maximal limit.
d. Attach the syringe to the other end of the plastic tubing.
e. Using a small graduated cylinder, add the required buffer volume directly to the
top of the column.
f. Reattach the columns top cap, and remove the bottom cap.
g. Drive the buffer through the column by pressing on the syringe, aiming for a drip
rate of 1 drip per second.
h. Once the syringe is fully depressed, wait until the column is barely dripping (no
pressure left in the column) and unscrew the top cap slowly. Withdraw the
plunger and reattach the top cap.
i. Repeat steps g and h until the required buffer volume has passed through
the column.
j. If you need to pause the procedure, attach the bottom cap to the column.
NOTE: If unscrew the top cap/disconnect the tubing while the column is still
pressurized, you risk passing air through your gel bed and/or spraying buffer on
yourself.
3. We will not be discarding our gels after use we will be saving them as
they are extremely expensive and reusable. To empty your column, add a
few mL of buffer, cap the column and rock back and forth until the beads
are fully suspended in the buffer. Then dump the contents of the column
INTO THE CORRECT RESIN BOTTLE. MIXING THE GELS WILL RUIN THE
ENTIRE BOTTLE. Repeat this procedure once more to collect residual
beads. Wash the column thoroughly with distilled water, attach both end
caps and return from where you got it.

82
Part I: Gel Filtration (size exclusion chromatography)
4) Mount a tall chromatography column on a retort stand using a clamp. Remove
the plastic cap from the bottom of the column, and place a waste beaker
underneath.
5) Fill the column with about 9 cm of Size Exclusion Chromatography Resin
(Sephacryl/Sephadex G25). The 9 cm refers to the height of the gel when it has
settled to the bottom of the column. This may require 10-15 mL (or more) of gel
slurry, so you may need to add multiple volumes of slurry to achieve this height.
To help the gel settle, you may need to apply pressure to the column as
described in the Note 2 above.
6) The next step is to equilibrate the gel with the mobile phase, which is our 50 mM
Tris buffer (pH 7.5). Obtain 100 mL of this buffer in a beaker or Erlenmeyer flask.
7) Add 20-25 mL of buffer to the top of the column.
8) Apply pressure to the column - aim for a rate of approximately 1 drip per second.
Stop when only a sliver of buffer remains above the gel surface.
9) With a P-1000 pipette, obtain 1 mL of the sample mixture from the instructor
(contains: Riboflavin, Catalase and Cytochrome C). Add the sample to the top of
your column.
10) Critical step (Read all before starting):
a. Prepare 20 mL of 50 mM Tris buffer (pH 7.5) in a small graduated
cylinder. Keep this handy for now.
b. Depress the plunger to apply slight pressure needed to fully embed the
sample into the gel (no liquid at top of gel).
c. Once embedded, immediately add the 20 mL of 50 mM Tris buffer (pH
7.5).
11) Obtain 6 small test tubes. Label one as Guide and the rest 1 through 5.
12) Fill the Guide tube with 3 mL of buffer. You will use this tube as a example of
the volume each of your fractions should be.
13) Hold tube 1 underneath the column and remove the bottom end cap. Apply slight
pressure to the column by depressing the plunger, achieving a drip rate of about
1 drip per second. Switch to tube 2 once the volume of tube 1 has reached
approximately that of the Guide tube. Continue with remaining tubes.
14) As you are eluting the products, try to identify qualitatively (by colour) when
Cytochrome C (red), Catalase (brown) and riboflavin (yellow) elute. Note you will
be trading your size exclusion column for an ion-exchange column (from another
group) for use in part 2.
15) ANALYSIS: This will be done in two parts - part a and b respectively.
a. Perform the Catalase test on each of the 5 test tubes as follows.
Catalase catalyzes the formation of water and oxygen gas from hydrogen
peroxide.
i. To test for the presence of catalase in a collected fraction, withdraw
50 L from each cuvette and place on a small square of parafilm. A
good strategy is to cut 5 small squares of parafilm about 1 cm 2 in
dimension.
ii. Using a dropper, add one drop of 10% hydrogen peroxide to each
of the fractions on the parafilm squares.

83
iii. Visually inspect the mixture for the formation of gas bubbles.
iv. Gas bubbles (O2 production) will indicate the presence of catalase.
v. Record your results in Table 1.
b. Measure the absorbance of each sample at 280 nm as follows:
i. Proteins are known to absorb UV radiation at 280 nm.
ii. Blank the UV-VIS spectrophotometer at 280 nm using 1 mL of Tris-
HCl buffer in a clean quartz cuvette.
iii. One by one, transfer approximately 1 mL of each collected fraction
to a clean quartz cuvette.
iv. Measure the absorbance of each sample at 280 nm.
v. Record the measured absorbance of each fraction at 280 nm in
Table 1.

Part II: Ion exchange chromatography


1) Obtain a small, empty chromatography column. Mount it on a retort stand using a
clamp. Ensure the bottom cap of the column is attached. Remove the top cap.
2) Obtain the bottle of Sepharose Cation Exchange resin, and withdraw
approximately 3 mL of slurry using a serological pipette. Add this to your column.
3) The goal is to achieve a bed height of approximately 1 cm, which may require
multiple volumes of resin slurry. Add volumes of resin slurry until the settled bed
height is approximately 1 cm. To drain excess buffer, place a waste beaker
underneath the column and remove the bottom cap. Allow the buffer to drip out
until sufficient buffer has cleared the column. Attach the bottom cap back onto
the column once the correct gel bed height has been reached. ALWAYS
ENSURE THERE IS BUFFER ABOVE THE TOP OF THE GEL.
4) Attach a segment of plastic tubing to the top cap of the column. Attach the top
cap to the column.
5) Obtain a 5 or 10 mL syringe. Pull out the plunger. Attach the other end of the
plastic tube to the tip of the syringe.
6) Follow Note 2 (above) to gently add pressure to the column. Continue until only
a sliver of buffer remains above the surface of the bed. Add the bottom cap back
onto the column.
7) Introduce 10 mL of 50 mM Tris-HCl buffer (pH = 7.5) to the column. You may
have to do this in 2 steps of 5 mL depending on the height of your gel bed.
8) Gently apply pressure to drive the buffer through the gel bed until only a sliver of
buffer remains over top of the gel bed.
9) Obtain a test tube rack, and fill it with 3 small test tubes. Label them 1 to 3.
10) Stand the first test tube so that the column is located just above it.
11) Obtain a P-1000 pipette and tip, and acquire 1 mL of sample mixture from the
instructor. Inject the sample mixture into the top of the column.
12) Remove the bottom cap, and apply gentle pressure with the syringe to embed
the sample into the gel. Once fully embedded, cap the bottom and quickly add 5
mL of 50 mM Tris-HCl buffer (pH 7.5).
13) Apply pressure to the column to drive buffer into Test Tube 1. Cap the bottom.
14) Introduce 5 mL of the Medium Salt Buffer (50 mM Tris, 50 mM NaCl, pH 7.5).

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15) Switch to Test Tube 2. Apply pressure to the column to drive the buffer through.
Attach the bottom cap.
16) Introduce 5 mL of the High Salt Buffer (50 mM Tris-HCl, 500 mM NaCl, pH 7.5).
17) Switch to Test Tube 3. Apply pressure to the column to drive the buffer through.
Cap the bottom. Note you will be trading your ion-exchange column for a size
exclusion column (from another group) for use in part 1.
18) ANALYSIS: This will be done is two parts - part a and b respectively.
a. Perform the Catalase test on each of the 3 test tubes as follows.
Catalase catalyzes the formation of water and oxygen gas from hydrogen
peroxide.
i. To test for the presence of catalase in a collected fraction, with
draw 50 L from each cuvette using a pipette and place on a small
square of parafilm. A good strategy is to cut 3 small squares of
parafilm about 1 cm2 in dimension.
ii. Using a dropper, add one drop of 10% hydrogen peroxide to each
of the fractions on the parafilm squares.
iii. Visually inspect the mixture for the formation of gas bubbles.
iv. Gas bubbles (O2 production) will indicate the presence of catalase.
v. Record your results in Table 2.
b. Measure the absorbance of each sample at 280 nm as follows:
i. Proteins are known to absorb UV radiation at 280 nm.
ii. Blank the UV-VIS spectrophotometer at 280 nm using 1 mL of Tris-
HCl buffer in a clean quartz cuvette.
iii. One by one, transfer approximately 1 mL of each collected fraction
to a clean quartz cuvette.
iv. Measure the absorbance of each sample at 280 nm.
v. Record the measured absorbance of each fraction at 280 nm in
Table 2.

NOTICE: Do not discard the gel resins it can be recovered for reuse. See
instructions at top of protocol.
Table 1:

Fraction # Catalase test (+/-) A280 nm

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Part II: Anion exchange
Table 2:

Fraction # Catalase test (+/-) A280nm

In-Class Assignment

Questions: Show instructor your answers before leaving GLP.

1. Which of your fractions from size-exclusion chromatography contained


catalase? Which contained Cytochrome C? What is the evidence backing your
claims?

2. Which of your fractions from size-exclusion possesses the most protein?


Which possesses the least protein?

3. Which of your fractions from ion-exclusion chromatography contain catalase?


Which fraction contained Cytochrome C? What can explain this segregation of
these two proteins?

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4. Which of your fractions from ion-exclusion chromatography contain the most
protein? Which possesses the least protein?

5. Are you able to determine which fractions (from both size exclusion and ion-
exchange chromatography) contain riboflavin? Explain your answer, and give
reasons for why riboflavin is present in these fractions.

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Laboratory 9 Milk Chemistry (2 week lab)
Objectives

1. Use the scientific method to solve problems

2. Use basic biochemical manipulations to separate the components of milk

- Casein

- Lactose

- Lipids

3. Draw conclusions that are supported by experimental data

4. Analyze data using common statistical measures

5. Apply your knowledge of the scientific method to real-life situations

Introduction

Most biochemical manipulations in the real world involve the separation of a complex biological
mixture into components, purifying them, and then identifying what you have. In some
instances, you may have manipulated the sample so that there are altered proteins, or there
may be an excess of carbohydrate but in the end, it is the job of the biochemist to separate
out those proteins, lipids, and small molecules and figure out what is there (qualitative analysis)
and how much of it is present (quantitative analysis).

The basic components of biological materials are water, protein, carbohydrates, fats and
nucleic acids. To learn about these components, we must first isolate them from biological
tissues. Although this can be a challenging process, one familiar biological substance is
relatively easy to analyse; milk. In this experiment you will separate the components of milk
and characterise them. At the end of the experiment, you should appreciate and be able to
assess the nutritional implications of milk components.

The separation of milk into its components for the production of butter, cheese and whey has
been carried out for many centuries. Most of the fat can be obtained simply by skimming the
layer that rises after standing, or by centrifuging. To make cheese, the major protein, casein, is
precipitated, together with the fat if it has not been removed. The carbohydrate, lactose,
remains in the whey, or filtrate, together with a mixture of salts, which includes about 12

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metallic cations plus chloride, phosphate and sulphate. A substantial amount of the phosphate
in milk is precipitated with the casein as phosphate esters of hydroxyl groups in the protein.

Isolation of the main components of homogenised milk in a laboratory procedure depends on


the totally different solubility behaviour of the three substances. The polypeptide chain in
casein contains more acidic than basic groups, and casein is soluble as a polymeric anion at the
pH of whole milk (about 6.0). The protein is very much less soluble at the isoelectric pH, 4.7,
where the anionic and cationic charges are balanced, so the addition of a small amount of
acetic acid to milk causes the casein to precipitate. The colloidal particles of butterfat, which
were kept in suspension by the presence of protein, agglomerate and separate with the casein.

After precipitation, the mixture is strained and the insoluble curd is extracted with acetone and
ether. In this step, the acetone acts as a co-solvent for both water and ether so that the fat
globules dispersed in the curd can be dissolved in the ether. The extract is then evaporated
completely to remove the solvent, and the fat is isolated. The recovery of butterfat by this
solvent extraction procedure is not complete, since some fat remains trapped in the casein.

The carbohydrate in milk is almost exclusively one sugar - the disaccharide lactose. The aqueous
whey contains a small amount of soluble proteins, lactalbumin and globulins, and these must
be removed before the lactose is isolated. In the laboratory procedure, the protein is
precipitated by diluting the whey with 9 volumes of ethyl alcohol. After removal of the protein,
the lactose crystallises directly in nearly pure form from the alcohol solution.

An example of the flow chart for this procedure:

Milk

Insoluble Soluble
Acetic Acid

Lipid + Lactose +
Trace Proteins
Casein

Insoluble Soluble Insoluble Soluble

Ether Ethanol

Casein Lipid Trace Lactose


Proteins

Recrystalize

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Laboratory Materials:

Whole Fat Milk


Measuring Cylinder
Beakers/flasks/test tubes
Watch/timer
Thermometer
Hot plate
Funnel/filter paper/cheese cloth

Table 1. Reagents

Reagent Handling PPE* Disposal

Acetic Acid - Wear appropriate PPE*. - Lab coat, nitrile - DO NOT dispose
gloves and of any reagents
- Wash thoroughly after Biotech/WWT down the drain.
handling. Laboratory approved
safety glasses** must
- Refer to MSDS be used for all
reagents and -Consult with
protocols used in this Professor for
Ethanol Flammable: Handle in lab.
Fumehood ONLY proper waste
- When splashes are a disposal.
concern,
Diethyl Ether Biotech/WWT
- Wear appropriate PPE*. Laboratory approved
safety goggles** must
- Wash thoroughly after be used by all
Acetone handling. members present in
the laboratory.
- Refer to MSDS

Anhydrous Sodium - Wear appropriate PPE*.


Sulfate
- Wash thoroughly after
handling.

- Refer to MSDS

*PPE (personal protective equipment)

** Refer to Laboratory Safety Manual

90
Protocol

Week 1: Part 1 - Acid treatment of milk

1. Place 50 mL of homogenized fresh milk in a 250 ml conical flask and warm on a hot plate until
40 C.

2. Using a plastic dropper, add about 1 mL of 50% acetic acid to the milk, swirling after every
few drops, until a curdy precipitate forms. Allow the mixture to cool while the curd coagulates
for 5 minutes.

3. Fold a 30 cm square piece of cheese cloth into a 10 to 15 cm square pad of several


thicknesses, place the cloth over a 250 ml beaker. Using masking tape, fasten the cloth to the
beaker. Slowly pour the coagulated milk through the cloth. Squeeze the gummy mass firmly.

4. Transfer the filtrate (whey) to a measuring cylinder and record the volume.

5. Transfer the solid in the cheese cloth back to the conical flask, scraping as much material as
possible from the cloth, for isolation of butterfat and casein.

Week 1: Part 2 - Isolation of Lactose

1. Place 10 mL of the cloudy whey solution into a 250 mL conical flask and add 100 ml of
ethanol.

2. Using your hot plate, warm the solution until it reaches 55 oC and let stand for 3 minutes.
Notice the appearance of precipitate. Filter by gravity through fluted filter (see demonstrator)
placed inside a plastic funnel into a 250 mL conical flask.

3. If the initial filtrate is turbid, pour it back into the funnel; the filtrate should be free of all
suspended solid. The filtration will take some time to complete since the precipitated protein
gradually clogs the filter.

4. When the filtration is complete, cork the flask and allow it to stand undisturbed. Set the flask
aside for the week.

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Week 1: Part 3 - Isolation of Butterfat

NOTE - All manipulations using ether must be done inside the fumehood!

1. To the milk solid in the conical flask from above (Part 1), add 10 mL of acetone and 10 mL of
ether.

2. Stir the mixture thoroughly, smoothing out lumps with a rubber policeman.

3. Pour off the solvent layer into a boiling tube.

4. Add 15 mL of ether to the solid again, stir and pour into the same boiling tube.

5. Repeat with an additional 10 mL of ether.

6. Evaporate the ether solution gently by placing it in a beaker of water in the fume hood. The
water in the beaker should approximately 40oC (on a hot plate). Allow the solvent to evaporate
until the residue is a clear yellow oil plus a cloudy water layer.

Week 1: Part 4 - Isolation of Casein

1. To the solid remaining in the conical flask after ether extraction (from Part 3), add 15 to 20
mL of acetone.

2. Stir and smooth lumps. Then pour the acetone into a Buchner funnel set up for suction
filtration. Be sure to place a small filter paper in the Buchner funnel

3. Stir the solid with another 20 mL of acetone, rub well and then collect the solid on the
funnel.

4. Place the filter paper containing your product in a small beaker and allow to dry over the
week.

WEEK 2

Week 2: Part 1 Isolation of Lactose

1. Obtain the conical flask containing your Lactose crystals from last week (Week 1 Part 2).

2. Without disturbing the crystals at the bottom, use a serological pipette to remove 50 mL of
the ethanol and transfer it to a new beaker.

3. Scrape the crystals loose and collect by suction filtration on a Buchner funnel.

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4. Use portions of the saved ethanol to rinse the remaining crystals into the funnel.

3. Remove the filter from the Buchner funnel and allow to air dry.

4. Record the weight of the lactose.

5. Knowing the original volume of the whey (Week 1 Part 1), calculate the weight that would
have been obtained from the entire sample.

Week 2: Part 2 Isolation of Butterfat

1. In the fume hood, identify your boiling tube from last week (Week 1 Part 3), and add 5 mL of
ether to the oily residue. Swirl briefly.

2. Obtain a new boiling tube, note the weight.

3. Place the boiling tube in a rack under the provided funnel (fume hood).

4. Press a cotton wad into the base of the funnel.

5. Add half a teaspoon of anhydrous sodium sulfate on top of the cotton.

6. Pour your ether/oil sample onto the center of the column.

7. Using a serological pipette, add (drop-wise) 3 mL of ether to the cotton swab to elute the
sample into the new boiling tube. Do not do this too slowly or the ether will evaporate from the
cotton.

8. Repeat with another 3 mL of ether. Repeat again if necessary to drive the yellow oil solution
through the cotton.

9. Boil-off ether layer in warm water bath until only oil remains.

10. Congeal oil in an ice bath in the fume hood.

11. Check for residual ether on top of oil layer. If yes, lay tube on its side until ether evaporates.

12. Weigh tube and calculate weight of butterfat yield.

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Week 2: Part 3 - Isolation of Casein

1. Obtain and record the weight of an empty weigh boat.

2. Collect the solid from Week 1 Part 4 onto the weigh boat.

3. Measure the weight of your protein.

Calculations + Questions GLP:

Provide the values for the following:

1. Total Volume of Milk: ______________

2. Total Volume of Filtrate (after acid treatment): ____________

3. Total fat content (grams): _______________

4. Weight of Lactose in Total Sample (grams): _____________

5. Weight of Casein (grams): ______________

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In-Class Assignment

1. For those measurements that are provided by the manufacturer, how do your measurements
compare to those of the factory? (refer to the nutrition label of the milk bottle)

2. Why do we use ether for the fat extraction?

3. What is sodium sulfate used for in the fat isolation?

4. What else might you want to know about milk that we didnt analyse here? How might you
measure those components?

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