CHEM 161L Biochemistry Laboratory 2

1st Quarter SY 2010-2011

Protein Characterization by Electrophoresis

Solidum, Andrew .1, Chan, Catherine T.2

1
Professor, School of Chemical Engineering, Chemistry and Biotechnology, Mapua Institute of Technology; 2Student, CHM161L / C20, School of Chemical Engineering,
Chemistry and Biotechnology, Mapua Institute of Technology

ABSTRACT

Another type of electrophoresis is SDS-PAGE or sodium dodecyl sulphate-polyacrylamide gel electrophoresis. PAGE is commonly used
for the separation and qualitative analysis of protein. Agarose can also be used but polyacrylamide gel can give better resolution
compared with agarose. Protein has different charge depending on the amino acid comprises the polypeptide chain thus, when
subjected to polyacrylamide gel electrophoresis, the separation will be based on the charge of the protein and its size. In SDS-PAGE,
SDS denatures the protein into its subunits and with an excess, it coats the protein with negative charge. With this, the protein will be
separated just according to its size since they all have the same charge. This is the advantage of SDS-PAGE compared to PAGE. In the
experiment, bovine serum albumin (BSA) should be subjected to SDS-PAGE for protein characterization but problem arises due to the
gel not able to polymerize. The experiment was not able to perform. A possible source of error is the shelf life of the reagents used
(agarose, APS). The goal of the experiment is to familiarize the students to the principle of SDS-PAGE and to characterize the protein,
BSA, into its subunits.

Keywords: albumin, casein, invertase, Bradford Assay, Warburg-Christian Assay, Benedict’s reagent

INTRODUCTION

Electrophoresis is one of the techniques that qualitatively
analyse protein sample or mixture. This technique is use in
biochemistry as well as molecular biology. Biomolecules
such as DNA and protein are separated based on the
electrophoretic mobility of the molecule dependent on the
size, charge, shape and chemical composition. Type of
electrophoresis that is commonly used for protein is
polyacrylamide gel electrophoresis. Other type of
electrophoresis, agarose gel electrophoresis, can also be
used but the resolution of the bands or the separation of
protein is much better when PAGE (polyacrylamide gel
electrophoresis) is used.
Commonly use PAGE is SDS-PAGE or sodium dodecyl
sulphide-polyacrylamide gel electrophoresis where, the
basis of protein separation is solely dependent on size.
SDS-PAGE is one of the denaturing electrophoretic
techniques used. Sodium dodecyl sulphate, SDS,
denatures the protein into its subunit (polypeptide
components). In an excess amount, SDS will make coat the
protein with negative charge thus, proteins in sample will
have the same charge and the separation will be based on
the size of the protein. Cathode, negative electrode, is
placed on top and anode, positive electrode, is placed at
the bottom of the gel. As the current is applied, the running
buffer provides ion to carry the current. The cathode’s
negative charge and the protein coated to be negatively
charge will repel and go through the positive side (anode)
and this is how the protein moves through the gel. Smaller
molecule will have smaller friction with the gel so it will
travel faster compared with the larger molecule.
The SDS-PAGE electrophoresis gel is comprised of two,
the stacking gel and the resolving gel. The stacking gel has
larger pores compared to the resolving gel. This helps in
the process of packing the protein so they will enter into the
resolving gel all at the same time. In the stacking gel, the
spreading of the sample was decreased before the protein
micelles enter the resolving gel. The size of the gel’s pore
can be changed depending on the protein sample. The
higher the per cent of the gel, the smaller the gel’s pore will
be. If the protein to be analysed or purify is large, the per
cent gel should be lower.
There are two advantages of SDS-PAGE to PAGE and
these are: (1) it coats polypeptides with negative charge so
it will migrate towards the anode and (2) it masks the
natural charge of the polypeptide so they will separate
according to their molecular size.

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 CHEM 161L Biochemistry Laboratory 2
1st Quarter SY 2010-2011
The mobility of the biomolecule is directly proportional to
the log of the corresponding molecular weight. The mobility
is expressed as the effective mobility or Rf (retention factor).
The Rf is the distance migrated by the protein/ distance
travelled by the tracking dye. Having the R f of the marker
and its corresponding log of molecular size, this serves as
the standard to know the size of the protein sample to be
analysed thus, electrophoresis (SDS-PAGE) is also an
analytical method.
This study’s objective is to familiarize the students to how
the principle of electrophoresis and also, to characterize the
given protein sample (i.e. subunits comprises the sample,
the molecular weight, etc.), BSA (bovine serum albumin).
MATERIALS AND METHODS
Running Gel Preparation
The following reagents were mixed to made the 7.5% gel
with 9.2 mL total volume using micro pipette: 1.75 mL 40%
stock acrylamide, 0.925 mL 2% stock bisacrylamide, 1.75
mL 2M Tris-HCl pH 8.8 and 4.9 mL distilled water. After, the
solution was degas for ten minutes using vacuum aspirator.
To activate the running gel, the following reagents were
added: 94 µL 10% SDS, 62.5 µL freshly prepared 10%
APS and 1.6 µL TEMED. The gel mixture was swirl not
more than three times Using 1 mL pipetor, the gel mixture
was transferred in the casting tray up to 1 cm below the
level of the comb. The gel was overlaid with small amount
of isobutanol-water solution. The mixture was allowed to
polymerize and the isobutanol layer was then removed by
washing the gel with distilled water.
Stacking Gel Preparation
The following reagents were mixed to made the stacking
gel: 0.6 mL 40% stock acrylamide, 0.28 mL 2% stock
bisacrylamide, 0.70 mL 0.625 M Tris-HCl pH 6.8 and 1.88
mL distilled deionized water. Before pouring the gel in the
glass plate, 50 µL 10% SDS, 120 µL freshly prepared APS
and 3.8 µL TEMED were added to the mixture for it to be
activated. The mixture was poured over the polymerized
resolving gel using pipette. The comb was placed on top
and there should be no air bubble. The gel was allowed to
polymerize. The comb was then removed (in one fluid
motion).
Loading Buffer and Sample Preparatiton
Experiment 02│ Group 1│ September 13, 2010
The loading buffer was prepared by combining the following
and diluted to 25 mL distilled water: 2.5 mL 10% SDS, 2.5
mL 0.625 M Tris-HCl pH6.8, 2.5 mL 10% glycerol, 5.0 g
0.02% bromophenol blue and 12.0 g 8 M urea.
100 µL of BSA (Bovine Serum Albumin) was transferred in
a micro test tube using pipette. 100 µL of the prepared
loading buffer was then added. The sample was placed in a
boiling water bath for ten minutes and immediately
immersed in an ice-water bath for three minutes.
Sample Loaidng and Gel Running
First, running buffer was prepared by mixing the following
and diluting to 1 L: 3.0 g Tris base, 14.4 g glycine and 1.0 g
SDS. The polymerized gel slabs was placed in the gel
chamber. The chamber was then filled with the running
buffer (the gel was completely immersed). The wells were
rinsed with buffer. The air bubbles at the bottom of the gel
were removed. The electrophoretic set-up was placed on a
level surface. The sample was loaded into the wells using
micropipette, 5-10 µL. The voltage was asset to 100 volts.
The protective electrode covering was then placed and the
gel was run until the dye reaches a 1 cm level from the
bottom of the gel slab.
Staining and Visualization
Staining solution was prepared by mixing the following and
diluted it to 100 mL using 100-mL volumetric flask: 50 mL
methanol, 10 mL glacial acetic acid and 0.25 mg Coomasie
brilliant blue (R250). The glass plate was transferred from
the gel apparatus to a flat-bottom container. Small amount
of staining solution was added to completely immerse the
gel plate. The gel from the glass plates was removed using
a spatula and incubated in the staining solution with mild
shaking until the desired band intensity is observed. The
destaining solution was prepared by mixing 25 mL 95%
ethanol and 5 mL glacial acetic acid and diluted it to 100 mL
distilled water using 100-mL volumetric flask. The
background staining was removed by several washings of
destaining solution with vigorous shaking until the bands
are visible. The gel was then dried under cellophane.
RESULTS
The experiment was unable to proceed to the
electrophoresis proper. The stacking and resolving gel were
unable to polymerize.
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 CHEM 161L Biochemistry Laboratory 2
1st Quarter SY 2010-2011

DISCUSSION

Polyacrylamide gel is made by co-polymerization of

acrylamide monomer (40% stock acrylamide) and the N, N-
methylene bisacrylamide (stock bisacrylamide), which is a
cross linking agent. APS (ammonium persulphate) and
TEMED (tetramethylenediamine) was added just before the
gel was ready to polymerize. This is because TEMED
catalyzes the crosslinking of the acrylamide monomer and
the N, N-methylene bisacrylamide and APS initiates that
reaction. SDS denatures the protein to its subunits and
coats the protein with negative charge.

The stacking gel and resolving gel were unable to

polymerize and possible sources of error are: the stock
acrylamide used was not newly obtained and its shelf life is
approximately three years. It might be that its shelf life is
already met thus, it is not usable anymore; also, the APS
might be the problem. Its shelf life is only one year with
good storage and this is shorter compared to the
acrylamide. Thus, even if the APS is freshly prepared, it is
still not able to initiate the crosslinking of acrylamide
monomer and bisacrylamide. APS is unstable. That’s why
after a week, the prepared APS cannot be use anymore.

REFERENCES

            1. Weaver, Robert F. (2001) Molecular Biology, 2nd

Edition

            2. Cox, Michael M., Nelson, David L. (2005)

Lehninger Principles of Biochemistry, 4th Edtion.

            1. Garrett, Reginald H., Grisham, Charles M. (1999)

Biochemistry, 2nd Edition

            2. Murray, Robert K., Granner, Daryl ., Mayes, Peter

A., Victor, Rodwell W. (2003) Harper’s Illustrated
Biochemistry, 26th Edition

            1. Weaver, Robert F. (2001) Molecular Biology, 2nd

Edition

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