LABORATORY REPORT

SBT 1043

BIOTECHNOLOGY CONCEPTS

AND TECHNIQUES

EXPERIMENT 3: PROTEIN

By

Bil. Name Student ID Lecture

Group
1 NUR ILYANA BT AHMAD E20172019103 A
RAZALI
2 AIREEN SHAZWANI BT E20172019109 A
SHAHARUDDIN
3 ROHADATUL ANNIS BT E20172019110 A
ZOLMI
4 MUHAMAD HASRUL E20172019097 A
AKMAL BIN ABDUL HALIM

LECTURER NAME:

DR. NURHAIDA BINTI KAMARUDDIN

OBJECTIVE:

PART A: Crude Escherichia Coli protein extraction

-To isolate crude protein from Escherichia coli using sample buffer

PART B: Preparation of separation/resolving gel and stacking gel

-To prepare SDS-polyacrylamide gel

PART C: Running SDS-PAGE

-To preform protein separation through SDS-PAGE
INTRODUCTION:

Proteins are polymers of amino acids. Each amino acid contains a central carbon,
hydrogen, and a carboxyl group, an amino group, and variable R group. The R group
specifies which class of amino acids it belongs to: electrically charged hydrophilic
side chains, polar but uncharged side chains, nonpolar hydrophobic side chain, and
special case.

Proteins have a variety of function in cells. Major functions include acting as

enzyme, receptors, transport molecules, regulatory proteins for gene expression, and
so on. Protein is one of the most abundant organic molecules in living systems and
has the most diverse range of functions of all macromolecules. Each cell in a living
system may contain thousands of proteins, each with a unique function. Their
structures and their functions are very greatly. They are all, however, polymers of
amino acids arranged in a linear sequence.

We run this experiment by using Escherichia Coli. Escherichia coli are one of the
organisms of choice for the production of recombinant proteins. Its use as a cell
factory is well-established and it has become the most popular expression platform.
For this reason, there are many molecular tools and protocols at hand for the high-
level production of heterologous proteins, such as a vast catalogue of expression
plasmids, a great number of engineered strains and many cultivation strategies. We
review the different approaches for the synthesis of recombinant proteins in E. coli
and discuss recent progress in this ever-growing field.
METHOD:

*refer jotter
RESULT:

Figure 1 shows proteins that have been loaded into the SDS-PAGE gel wells before running
at 100v. From the most left is the protein ruler and the other wells are protein samples.

Figure 2 shows the PageRuler™ Prestained Protein Ladder, 10 to 180 kDa that was
used as reference of size standards in protein electrophoresis.
 Our group’s result

Figure 3 shows the result of protein samples after running at 100v and after the dye is
distained overnight.
DISCUSSION:

PART A: Crude Escherichia Coli protein extraction

For part A, we isolated crude protein form Escherichia coli using sample buffer.
Before added sample buffer, we completely removed any residual of liquid media by
pipetting. After that, 100µƖ of sample buffer was added to the cell pellet. This sample
buffer is used for the preparation and loading of protein samples onto a gel for SDS-
PAGE analysis. SDS contained in the sample buffer is used to denature protein and
make them negatively charged. Beta mercaptoethanol is used to break disulphide
bonds. Glycerol increases the density of the sample relative to the surrounding
running buffer making it easier to load in the well. Besides that, sample buffer
contained Bromophenol blue is used to follow the run of protein sample on the gel.
After inserted sample buffer, it must be heated at 95℃ for 10 minutes and placed it on
ice box to maintain the temperature.

PART B: Preparation of separation/resolving gel and stacking gel

1. Preparation of separating gel (12%) for SDS-PAGE:

dH2O 1.7 mL
Acrylamide 30% 2.0 mL
Tris1.5M (pH 8.8) 1.25 mL
SDS 10% 25 µL
Ammonium persulphate 10% (freshly prepared) 25 µL
TEMED 2.5 µL
Total 5 mL

2. Preparation of stacking gel (5%) for SDS-PAGE:

dH2O 1.7 mL
Acrylamide 30% 2.0 mL
Tris0.5M (pH 6.8) 1.25 mL
SDS 10% 25 µL
Ammonium persulphate 10% (freshly prepared) 25 µL
TEMED 2.5 µL
Total 5 mL
 Separating gel is a group of protein which when it touches the resolving gel, it can increase
the voltage of SDS-PAGE that will affect the protein separation by size as they move down
through the small pores. While stacking gel act as to stacks all the protein from the sample as
narrow as possible in a band form in order for the protein to enter the resolving gel at the
same time because the gel is low in pH and acrylamide percentage which is why stacking gel
is placed on top of separating gel. The difference between separating and stacking gel are two
different concentration and pH value of Tris for better protein molecule migration and the
sizes of pores of stacking gel is bigger than separating gel.

In the gel mixture, Acrylamide 30% is used to separate the protein fragments in small bp
based on their densities. The reason why there are 2 different pH is because it is charged
negatively at higher pH. When the power in the running buffer goes on the glycine ions, they
want to move away from the cathode (the negative electrode) so that they head towards the
sample and the stacking gel. The pH is low, so they lose a lot of their load and slow down.

The function of Sodium Dodecyl Sulfate (SDS) is used to isolate and identify protein
molecule by the help electrophoretic system. Ammonium Persulfate (APS) is an oxidizing
agent that is used with TEMED that induces for polymerization of acrylamide and form
mesh. Thermo Scientific Pierce Tetramethylethylenediamine (TEMED) acts as catalyst that
help APS for polymerization.

After all the separating gel mixture is done, the mixture then poured into the casing frame.
Followed by the stacking gel after the separating gel is hardened. The comb is put on into the
stacking gel to create the well to load the protein samples.
PART C: Running on SDS-PAGE

SDS-PAGE is a very common laboratory technique used to analyze proteins. The acronym
SDS-PAGE stands for Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis.
Sodium dodecyl Sulfate or SDS is a detergent commonly used in biology laboratories to
denature proteins. Polyacrylamide is a polymer prepared as a gelatinous medium or “gel”
through which proteins can be resolved based on molecular weight. Electrophoresis refers to
the movement of charged soluble particles such as proteins through a medium during
exposure to an electric field. SDS-PAGE is therefore a technique by which proteins move
through a polyacrylamide gel that is subjected to electric current.

The figure shows an illustration of an apparatus used for SDS PAGE.

In the process of running the SDS PAGE, we denatured and loaded the several different
protein samples on a polyacrylamide gel and then the gel was run as described. Preparing an
acrylamide gel for SDS-PAGE is a bit tricky, so the polyacrylamide gels have been prepared
for each group. To denature the proteins it is essential that we add sample loading buffer and
then boil the sample. The sample loading buffer contains several key components needed to
fully denature the proteins and have them run through the gel properly. We loaded the
samples into the wells really carefully to make sure not to damage the size of the wells or not
to pour the sample out of the well instead of pouring inside it. At this stage, sample of the
proteins appears to be blue because of a dye (bromophenol) used while preparing the sample.
Even the pipet tip is too big to be inserting directly into the well. Before you load your
protein samples you will load a practice sample so you will have some experience before
loading your real samples. A 150 voltage is applied after dipping the “sandwich of gel and
glass plates” in running buffer. We turn of the voltage when the tracking dye has reached or
crossed the gel. The gel is further preceded for the subsequent analysis.

CONLUSION:

In this experiment we manage to isolate crude protein from overnight grown Escherichia
coli by using a sample buffer to be further use in running SDS-PAGE. We also manage to
prepare two types of the SDS-polyacrylamide gel which is white odourless and soluble in
water gel, according to the recipe that have being given in the lab manual which is separating
gel and stacking gel. Lastly we achieved to perform protein separation through SDS-PAGE
method using SDS-PAGE apparatus by loaded the sample protein from previous step into the
well and run the SDS-PAGE at 150 volt for 30 minute.
REFERENCES:

1. Polymenis, M. (Ed.). (2010, September 26). SDS-PAGE stacking gel - BICH 608
(Polymenis-Pettigrew). Retrieved May 8, 2019, from
https://sites.google.com/a/tamu.edu/bich608-602/bich608---announcements-and-
more/sds-pagestackinggel

2.SDS Page Gel Electrophoresis. (2001, May 1). Retrieved May 8, 2019, from
https://ww2.chemistry.gatech.edu/~lw26/course_Information/4581/techniques/gel_ele
ct/page_protein.html

3. Ali. (2019, January 13). Retrieved May 8, 2019 from https://howbiotech.com/the-

principle-and-procedure-of-polyacrylamide-gel-electrophoresis-sds-page/