Delfino E.

Castelan

BITC 2411

Jose Miguel Arriaga, M.S.

Polyacrylamide Gel Electrophoresis

Introduction: In this experiment we determined the molecular weight or predetermined

proteins at know concentrations by means of polyacrylamide electrophoresis. The technique used
was SDS-PAGE, Sodium Dodecyl Sulfate (SDS), also known as sodium laurel sulfate, is an
anionic detergent that binds to proteins and disrupts non-covalent bonds of proteins and converts
then to negatively charged molecules so that all will migrate towards the anode. Polyacrylamide
is a very commonly used matrix for electrophoresis, run vertically, and is much thinner than
agarose gels. The Buffer system used in this experiment was discontinuous because two types of
gels were used; a second stacking gel was placed on top of the resolving gel. In the Laemmli
system, different buffers are used in the stacking and resolving gels. Both the pH and the
concentration of the components are different. These buffers are also different from the tank
buffer in both concentrations and pH. There were two particularly interesting features to this
discontinuous system. One was that the dilute protein sample in the well is concentrated at the
interference of the stacking and resolving gels before entering the resolving (running) gel. The
second feature is very interesting to mention but was not applied in this experiment, and it is the
ability to resolve proteins into readily distinguishable and relatively narrow protein bands that
are readily cut out; and once removed, the protein band may be injected as an antigen or micro
sequenced.

Materials and Methods: The different types of proteins to be diluted were taken from
stock, Bovine Serum Albumin, Bovine IgG, Bovine Serum, and two prestained SDS-PAGE
standards: Low Range (18,500-106,000 Daltons) Cat #161-0305, and High Range (49,500-
205,000 Daltons) Cat #161-0309 from Bio-Rad®. Gel Casting Supplies, Glass plates (2, thin and
thick), a gasket to support, comb, side-arm flasks, beakers 50mL-1L, graduated pipettes,
micropipettes 5-200µL, analytical balances (to weight chemicals), weight dishes, Mohr pipettes,
loading pipettes, Mini-PROTEAN 3 electrophoresis chambers, and a PowerPac Basic power
supply. From the chemicals used, HCL diluted 50%, Tris-HCL buffer from powder formula wt.
121.14, Tris Base, SDS, Ammonium Persulfate, Running Gel Buffer, Monomer Acrylamide
Solution, dithiothreitol (DTT), glycerol, bromphenol blue, glycine, and distilled water. Also used
were premade solutions for fixative, isopropanol, acetic acid, and dye solution Coomassie
Brilliant Blue G-250 with methanol and water, along with distaining solution of acetic acid and
methanol.

The methods employed are found in the laboratory guide provided. First, the vertical gel
apparatus was assembled and was tested with water to see if there were any leaks, until it was
ready for the acrylamide. The focused of the first step was on preparing stock solutions. The 4X
Running Gel Buffer was made in a 150mL beaker by adding 1.5M Tris-HCl, pH 8.8 and it was
adjusted by using 5 molar HCl starting from pH 11.1 down to the desired solution and diluted in
a 100mL volumetric flask with distilled water. The 4X Stacking Gel Buffer was made with the
same Tris-HCl combination but had a pH of 6.8 and a volume of 50mL when completed. A 10%
SDS solution was made by mixing stock solution to the final dilution of 50%. 1mL of
Ammonium Persulfate was made from stock. Running Gel Overlay Buffer was prepare by
adding 25mL of Running Buffer and 1mL of 10% SDS solution and diluted to 100mL
volumetric flask with distilled water. The 2X Treatment Buffer was made by adding 2.5mL of
Stacking Buffer, 4mL of 10% SDS, 2mL of Glycerol, 2mg of bromphenol blue, .31g DTT and
enough distilled water to bring the volume to 10mL. Then it was aliquot in .5mL vials and stored
in the -20ºC. Tank Buffer was prepared by adding 3.03g Tris Base, 14.41g glycine and 1g SDS
to 800mL of distilled water, then brought to 1L volume before storing in glass bottles enough to
contain.

After the mini vertical cell electrophoresis chamber had been assembled and sealed and
tested for leaks, the resolving gel was prepared in a 125mL side-arm flask. 5mL of Acrylamide
Monomer, 5mL of Running Buffer, .2mL 10% SDS, and 9.7 distilled water. It was degassed for
5 minutes with a vacuum pump while stirring and then two remaining ingredients were added
before pouring the gel, 100µL ammonium persulfate, and 6.7µL TEMED. The solution was
added between the two glass plated using a glass Pasteur pipette at an angle until the level was
close to the comb by .5cm. The excess acrylamide solution was saved to watch the
polymerization timeframe. A deep layer of water saturated n-Butanol was added on top of the gel
to avoid evaporation and then it was put into a 35ºC oven and left overnight covered with
parafilm. The stacking gel was then prepared in a 50mL side arm flask. Similar to the previous
step, acrylamide monomer was added (0.8 mL), 4X stacking buffer (1.66mL), 66µL of 10% SDS
solution and 4.06mL of distilled water. After degassing for 5 minutes while stirring, the two
other ingredients were added; 33.4µL of ammonium persulfate and 3.3µL of TEMED, and it was
gently stirred before being added to the vertical plates. The remaining butanol was removed from
the top of the resolving gel and the stacking gel was added on top of it until the plates were
filled, then the comb was added and then the gel was covered with plastic and left overnight to
polymerize.

For the loading step, various dilutions of proteins from stock were made, and loaded in
enough quantities to each lane so that the results were clear and have a high resolution.
Depending on the thickness of the gel (.25 cm in this case), the purity of the protein and the
sensitivity of the protein stain used .5-50μg of protein per lane may be loaded. The following
stock solutions were used: BSA, bovine serum, bovine IgG, each at 10 mg/ml. Then, they were
diluted according to dilution table 1.

Dilution Table 1. Preparation Samples from stock

Protein Sample Desired Dilution Stock Solution Diluent (Buffer) Total 2X Load

BSA and Serum 1 µg / 5 µl (.2) 10 µg / µl .4µl +19.6 Buffer 20 µL

IgG 1 µg / µl 4µl + 16µl

BSA and Serum 3 µg / 5 µl (.6) 10 µg / µl 1.2µl + 18.8µl 20 µL

IgG 1 µg / µl 12µl + 18µl

BSA and Serum 10 µg / 5 µl (2) 10 µg / µl 4µl + 16µl 20 µL

The samples were diluted in running buffer to obtain an overall negative charge from the
SDS detergent so that they would migrate toward the anode and be easier to distinguish them
from the molecular weight only and not from the relative charge on them. The different dilutions
were prepared according to Table 1 so that 20 µl were made for running two gels. The molecular
weight markers were premade and prestained in 33% (v/v) glycerol, 3% SDS, 10mM Tris pH
7.0, 10mM DTT, 2mM EDTA, and 0.01% NaN3. An equal volume of 2X treatment buffer from
material was added to the samples and then they were placed in a boiling water bath for 90
seconds so that the proteins could be denatured to its original linear structure. Finally the gel was
ready to be loaded according to the following diagram:

Table 2. SDS-PAGE Protein Loading Diagram

1 2 3 4 5 6 7 8 9 10

Low .2 .6 2 .2 .6 .2 .6 2 High
Marker BSA BSA BSA IgG IgG Serum Serum Serum Marker

The comb was removed from the gel, and the wells were rinsed with tank buffer to
remove any unpolymerized acrylamide that might interfere. The inner chamber was filled with
buffer to eliminate bubbles until the wells were filled. Then the protein samples were loaded
according to table 2 in 10μl samples to each well. The electrophoresis chamber was already
assembled so that the gels casted were just transferred to the chamber and tank buffer was added
so that the upper buffer chamber was in contact with the gel. The vertical gel set up was covered
and connected. The power supply was set at 200 volts. According to the protocol we should set
the voltage at 170 volts. “It’s possible to use higher voltages serving to shorten the time required
for the run but it also supplies more heat resulting in more problems. As these gels are not
cooled, keeping the voltage lower helps to reduce the heat load” (pg 8). The higher voltage did
produce greater heat but the buffer chamber also contained an ice pack to reduce the heat
produced by the current in the gel.

When the tracking gel approached the bottom of the gel, the power was turned off. The
approximate time the gels were run was 50 minutes. Then the apparatus was unplugged, opened,
and the gels were taken out, removed from the plates, and placed into fixative for 15 minutes.
For the staining procedure, the gels were transferred to a plastic tray filled with Coomassie
Brilliant Blue G-250 and left until the bands were visible, for about 20 minutes. To distain the
gels, they were placed into a gel distaining solution made of acetic acid and methanol and left
overnight to distain, then removed and dried for easier examination.

Safety: General safety rules were followed and some extra cautions were taken because
of the nature of the process and the materials used. Acrylamide is a skin irritant and a neurotoxin.
Because it may be absorbed through the skin and the fumes can be inhaled, gloves were worn at
all times, along with the lab coat as usual and the materials were always mixed under a fume
hood. Plenty of hazardous materials were present so the safety concerns were exacerbated this
time. The following chemicals known to posses hazards: acrylamide, SDS, APS, Temed, DTT,
and all biological material are also considered hazardous materials. The power sources used in
this experiment produce lethal currents, so extra precaution was taken when running the gels.
And the always present safety goggles were never forgotten of left out to protect such precious
sensory organs such the eyes.

Results and Discussion: A very clear gel was obtained after the whole process was
terminated. To examine the gel we used a scanned copy in print and measured the following
distances according to table 3 adapted from the laboratory protocol (pg 9). The bottom of the gel
was 55 mm and the marks were measured from the bottom of the gel to the middle of the band.
Resulting in a 55mm–the distance measured = distance migrated. The Table is in the Raw Data
form and in the corresponding section; the following is an interpretation of the data obtained
from extrapolating Rf values and Log10 of Molecular Weight to calculate actual molecular weight
of proteins and predicting the contents of each mixture.

Table 3. Examination of the PAGE Gel and estimation of MW

Low MW BSA BSA BSA IgG IgG Serum Serum Seru High MW
Marker m Marker

? ? 98000 Phospho-
93000 B 106000
 BSA
80000

Myosin Myosi Myosi Myosi Myosi Myosi Myosi ? Ovalbumi

205000 n n n n n n n 49500
19400 22000
0

Β-galc Β-galc B-galac B-gal Carbonic-

116500 12800 128000 1330 A 32500
0 00

BSA BSA BSA STI STI STI BSA STI STI STI 27500
80000 78000 2900 27500 2600 27500 2600

Lysozy Lysoz Lysoz Lysozy Lysozy Lysozyme

me yme yme me me 18500
17500 17000 18400 18500 17000

Ovalbu Ovalb Ovalbu Ovalb Ovalbu Ovalbu Oval

min umin min umin min min bumi
49500 49000 n

It’s clear that the samples not only contained BSA, Serum, or IgG but its constituent
proteins as well.

• Sample 1 from BSA had myosin, B-galactosidase, BSA, and Ovalbumin. Sample
2 from BSA detected that Lysozyme was present in the mixture as well. Sample 3
of BSA did not have either B-galactosidase or Lysozyme but the process detected
Soybean trypsin inhibitor, which is rare or it means that this protein is detected
only at low concentrations because it was the most diluted sample of BSA.

• Sample 1 of IgG also detected myosin, trypsin inhibitor, and lysozyme as well as
the second sample of IgG, which makes us wonder if any other protein could be
detected at lower concentrations because only two were used of this mixture.

• Sample 1 of Serum shows that it’s a very rich mixture of proteins as we found
myosin, BSA, lysozyme, and Ovalbumin, as well as two unknown proteins.
 Sample 2 shows even more variety including B-galactosidase, trypsin inhibitor,
lysozyme, ovalbumin, and two ‘apparently’ unknown proteins. In Sample 3 of
Serum we found no unknown proteins but the usual combination of B-
galactosidase, trypsin inhibitor, and Ovalbumin.

Looking at the standard curve for the high range proteins (graph 1) we see that the
first few points fell outside the standard range and unfortunately cannot be obtained from
the given formula. This explains the strange presence of unknown proteins which are,
according to the calculations and distance migrated, Phosphorylase-B in Samples 1 and 2
from serum and ovalbumin from sample 2 of serum respectively. There was no problem
with either points of formula from the low range molecular markers (graph 2), so the
marks and estimated numbers were easy to measure.

Graph 1. Standard Curve of High Range proteins including the formula.

Graph 2. Standard Curve of Low Range proteins including the formula.

Conclusion: The process of Sodium-Dodecyl Sulfate Gel Electrophoresis is very

effective in detecting proteins at very low concentrations. The results are very clear to analyze
and although the process in detailed it’s very convenient to use. The SDS really works and the
one-dimensional process of electrophoresis separated the proteins on the basis of molecular
weight only. The molecular markers are very useful to compare and make standard curves to
examine the rest of the bands after staining.

Raw Data and Calculations: Most of the calculations are simple arithmetic and are
found on the methods section of the reports. Most of the dilutions calculations are found in table
1 and 2, and some on table 3. The analysis of the molecular marker is also included in the data
for comparison and observation, noted as table 5.
 Table 4. Dye marks and the measured migrations from the origin.

Low MW BSA BSA BSA IgG IgG Serum Serum Serum High MW
Marker Marker

Distance: 10 9 9

Distance: 14

Distance: 15 15 15 15 15 15 20 20
15 mm

Distance: 25 25 24 28
25 mm

Distance: 37 37 33 34 35 37 34 35 34
37 mm

Distance: 43 44 42 44 44 44

Distance: 48 48 48 48 48 48
48 mm

MW vs. Rf Values of Proteins

Molecular Weight High Log Low
Protein Name (daltons) Range Rf MW Range Rf Log MW
Myosin 205000 0 5.312 0.273 5.312
β-galactosidase 116500 0 5.066 0.455 5.066
Phosphorylase B 106000 0.164 5.025 5.025
Bovine serum albumin 80000 0.255 4.903 0.673 4.903
Ovalbumin 49500 0.364 4.695 0.873 4.695
Carbonic anhydrase 32500 0.51 4.512 4.512
Soybean trypsin inhibitor 27500 0.618 4.44 4.44
Lysozyme 18500 0.8 4.267 4.267

Postlab Questions.
1. Yes, there are more bands found in both columns, these might be other protein in the
homogeneous mixture of proteins.

2. There are two bands because the molecule immunoglobulin G contains 4 fragments, 2 of the
same weight 53,000 and 22,500 respectively, for a total of 150,000 daltons.

3. There are many visible lines in the Serum lanes because it’s a rich mixture of proteins as noted
in Table 3.

4. 73 to 75000 daltons of molecular weight could be the marks that were attributed to the high
molecular weight markers but were really other proteins found in serum. Transferring could be
found at the very top of the gel which in fact has a line in two samples of serum.

The higher concentrations of proteins worked better such as the .2 and .6 µg/ µl but the low
concentrations worked as well which means the process of SDS-PAGE posses very high
resolution power for any protein mixture.