Separation of Proteins Part II

180

Agenda
Review Separation of Proteins Part I
What is SDS-PAGE and how does it work?
Western Blotting
Todays procedure
Reminders
Gel loading demo

What did you do two weeks ago?

What two proteins did you separate?

What two types of chromatography did you use to

separate the mixture of proteins?

What is SDS-PAGE
Sodium Dodecyl Sulfate-Polyacrylamide Gel

Electrophoresis

A method of separating proteins based on their electrophoretic

ability (length and mass-to-charge ratio)

What is SDS-PAGE used for?

Determination of protein size

Protein identification
Quantifying proteins
Blotting applications- Western blots

Preparing Samples for SDS-PAGE

Sample Preparation
Sample Buffer components:

Tris-Cl Buffer: maintains pH (~6.8)

SDS: sodium dodecyl sulfate, denaturing anionic detergent
-mercaptoethanol (BME): breaks disulfide bonds

YOU MUST WORK WITH BME IN THE HOOD!!!

Glycerol: increases sample density

Tracking dye (Bromophenol blue): helps track progress of
run

Add protein sample and heat buffer to 90C to

activate SDS and BME

Why is BME important?

BME breaks the disulfide bonds, removing tertiary and quaternary structure

What would happen if you did not add BME to your sample?

Why is SDS important?

SDS coats protein backbone with a negative charge
in a constant weight ratio of 1.4g SDS/ g protein
and linearizes the protein, removing secondary structure

SDS-PAGE Gel Set up

The SDS-PAGE Gel

4%

Acrylamide

Larger proteins are trapped by

the pores and travel less

20%

Smaller proteins fit through

the pores and travel farther

The gel is comprised of a gradient of polyacrylamide,

which helps to separate molecules by their size

How does SDS-PAGE work?

An electric field is
applied to the gel,
allowing proteins to
migrate by charge.
In which direction do
the proteins run and
why?
If there are multiple
bands in the same
lane, how would you
identify whether
it contains your
protein of interest?

Western Blotting
A method of detecting specific proteins by

transferring the proteins from a gel to PVDF

membrane and tagging the protein of interest with
antibodies
What is an antibody?
An antibody is a specialized

immune protein that recognizes

a specific target molecule

Todays Procedure
Add 75L of
Peak Fraction
1 to 15 L of
sample buffer

Repeat for
Peak
Fraction 2

Heat Sample
in heating
block (90C)
for 4 minutes

Centrifuge at
max speed for
30 sec.

Make sure that tubes all contain the

same volume and to properly
balance the centrifuge

Load samples
onto gel in the
correct order

Gel Loading Order

Please load 15L of sample and 10L of MW marker

E. coli Lysate
Today you will run lysate from

E. coli that over-express Galactosidase

These E. coli make an excess amount

of the enzyme that breaks down the
sugar -Galactose

E. coli are lysed to spill the

contents of the cells

When run on a gel, you will see the

total proteins expressed by the cell at
the time of lysis
Over-expressed proteins, will appear
darker than other bands on the gel
Western blotting allows for you to
determine if your protein of interest
is your lysate, when it is difficult to
determine from the gel

Setting up your Western Blot

Today, you will set

up your western
blot sandwiches:

An electric field will

be run through the
blotting apparatus,
which will transfer the
negatively charged
proteins from the gel
to the PVDF
membrane

Data Analysis
This Week:
Determine the protein concentration
using the Beer Lambert Law

Protein migration/dye front migration

Measure from the bottom of the well to the
dye front/protein band

Create a semi-log plot (use the

correct paper!)

MW vs Rf values for MW marker bands

Use standard curve to determine the
molecular weight of your peak fractions
Use the 2-cycle semi-log paper

(g/mol)

Next Week:
Calculate Rf values:

Standard curve of molecular

weights of proteins based on Rf
values

Reminders
Gloves and goggles
BME is toxic

You will find sample buffer in the hood

Please place all tips/tubes contaminated with sample buffer in the appropriately labeled
waste containers

Remember the order in which you set up your western blot

SAVE YOUR FRACTIONS FOR NEXT WEEK
Lab report for Parts II and III due week of 4/6
Problem Set 3 due the week of 3/30
Next week, we will:

Analyze the stained gel

Continue the western blot
Perform the Lowry Protein Assay (Separation Part III)

Reminders
Please remember to properly cite the documents

used in your concluding statements/discussions

http://classguides.lib.uconn.edu/content.php?
pid=352130&sid=3243107