Cell & Molecular Biology Protocols

Exercise 6. Analysis of Milk Proteins Using SDS-PAGE

In this exercise, proteins in solution will be separated by running them through a gel matrix subjected to an
electric field.

In electrophoresis, an electric field is applied to a solution of charged molecules that allows the molecules to
migrate at a rate depending on its size or charge. In sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE), denatured protein samples are run on an inert matrix composed of cross-linked polyacrylamide.
This separates the proteins in the solution by molecular weight:

Sodium dodecyl sulfate (SDS) is an anionic detergent used to denature and to mask the intrinsic charge of the
proteins so that they migrate solely based on their molecular weight. The SDS coats the proteins with an over-
all negative charge, allowing them to migrate towards the positive electrode. It also acts as a denaturing agent,
unfolding proteins by removing secondary, tertiary and quaternary structures formed by non-covalent
interactions between the components of the polypeptide backbone and the amino acid side chains. In the
presence of a reducing agent, such as β-mercaptoethanol, disulfide bonds formed between cysteine side
chains can also be broken. A solution of proteins of known molecular weight called a molecular weight marker
is run together with the samples and is used as a standard for determining the molecular weight of the sample.
Generally, the distance migrated by the protein is inversely proportional to its molecular weight (smaller
peptides migrate farther).

To visualize the proteins in a gel, the gel is stained (with Coomassie Brilliant Blue R-250 or silver). Proteins will
be visible as bands in the gel, with each band representing a protein of a specific molecular weight. The
molecular weight marker can be used to prepare a standard curve by graphing the log of the molecular weight
of each band versus its relative mobility (Rf). This can be used to determine the molecular weights of each
protein band of the samples loaded in the gel.

Pre-lab enrichment: Watch the reparation of an SDS polyacrylamide gel, sample prep, loading and running the
gel, and staining the gel:
https://www.youtube.com/watch?v=-H8GU_90Gak (FNH Teaching Lab)

Objectives:
At the end of the exercise, each student should be able to:
1. Perform SDS-PAGE and interpret the results.
2. Apply SDS-PAGE in characterizing proteins.
3. Use SDS-PAGE to separate and identify milk proteins.

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Materials:
Equipment/Glassware/Materials Reagents:
SDS PAGE electrophoresis Sample protein solution (Milk sample – example fat free, low fat,
apparatus whole or soy milk): protein concentration previously
Power supply determined by Bradford assay
Hot plate/heating block Distilled, deionized water
Rocker/orbital shaker For SDS-PAGE (see appendix for recipes)
25 ml glass beakers Acrylamide:bis monomer solution (40%, 37.5:1)
1.5 mL microcentrifuge tubes 4x Running Gel buffer
Micropipettors and micropipette 4x Stacking Gel Buffer
tips 10% SDS
Vortex 10% Ammonium persulfate
Ice buckets with ice Water saturated n-Butanol
Kimwipes Running gel overlay
Paper towels 2x sample treatment buffer
Tank buffer
TEMED (N,N,N´,N´-tetramethylethylenediamine)
For staining
Coomassie staining solution
Destaining solution

Procedure:

Preparation of SDS-polyacrylamide gel

1. Set up your gel casting apparatus (Bio-Rad Mini Protean® Tetra Cell)
2. Prepare the running gel.
Prepare the running gel monomer. Add APS and TEMED just prior to pouring the gel. Allow to
polymerize before adding stacking gel by overlaying gently with water or n-butanol.
Running Gel (7.5%) (recipe good for two gels)
Monomer solution 3.75 mL
Running gel Buffer 3.75 mL
10% SDS 150 µL
ddH2O 7.276 mL
10% APS 75 µL
TEMED 5.0 µL
3. Prepare the stacking gel.
After the separating gel has polymerized, decant the n-butanol and wash the gel with running gel
overlay. Prepare the stacking monomer, add the APS and TEMED and pour. Insert the comb and
allow to polymerize completely before running.
Stacking Gel (recipe good for two gels)
Monomer solution 665 µL
Stacking gel Buffer 1.25 mL
10% SDS 50 µL
ddH2O 3 mL
10% APS 25 µL
TEMED 2.5 µL

Sample preparation
1. Make three (3) ten-fold dilutions of your milk samples into 1.5 mL microcentrifuge tubes.
2. Dilute the samples 2:1 with the sample treatment buffer (i.e. 10 µL sample + 5 µL treatment buffer) in
a 1.5 mL microcentrifuge tube and heat at 95°C for 4 minutes prior to loading.

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Electrophoresis
1. Assemble the gels in the electrophoresis set up. Pour enough tank buffer into the chamber to
submerge the short plate.
2. Load 10 µL of each sample in separate wells. Load 5 µL protein molecular weight marker into a
separate well.
3. Fill the tank with tank buffer. Run the gel at a constant 200 V for 30 – 40 minutes (or until the dye
reaches the bottom of the gel).

Staining and destaining the gel with Coomassie

1. Carefully remove gel from glass plates. The stacking gel may be removed and disposed.
2. Transfer the gel to a staining pan with the Coomassie Brilliant Blue staining solution. Stain the gel for
4 hours to overnight with gentle rocking.
3. Transfer the gel to the destaining solution. Change the destaining solution every 30 minutes. Destain
until the background of the gel is almost transparent. Store the gel in distilled water and document.

Data analysis
1. Prepare a standard curve: Calculate the relative mobility (Rf) of each band in the standards (molecular
weight marker) and your samples by measuring the distance each band traveled from the top of the
separating gel, and then dividing this distance by the distance traveled by the dye front.
Migration distance of the protein
Rf =
Migration distance of the dye front
Plot the log MW of each protein in the standard versus its relative mobility.
2. Choose 5 of the most distinct protein bands in the milk sample and calculate the Rf value of each
band. Using the standard curve, determine the molecular weight of each band.

Record Results:

1. Photodocumentation of gel (label properly: number wells, indicate type sample loaded in wells):

Well Group/Sample
Number loaded
1
2
3
4
5
6
7
8
9
10

Molecular Weight Marker used: Bio-Rad Precision Plus Protein™ Standards

2. Preparation of standard curve:

Distance of dye front = ______________ mm

MW (Da) log MW distance travelled (mm) Rf

250,000

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150,000

100,000

75,000

50,000

37,000

25,000

15,000

10,000

Graph standard curve:

5.75

5.25
logMW

4.75

4.25

3.75
0 0.2 0.4 0.6 0.8 1
relative mobility (Rf)

Equation of the linear trendline from standard curve: _______________________

3. Identify 3 - 5 major bands in milk sample: measure the distance travelled of each band, calculate the
Rf, and using the standard curve, calculate the MW of each band.

Distance travelled
Sample protein band Rf Log MW MW (Da)
(mm)
1
2
3
4
5

Guide Questions:
1. What are major protein components of milk? What is the most abundant form of protein in milk and
what is its molecular weight?
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2. Do the molecular weights of the sample protein bands in the gel correspond to the major protein
components of milk?
3. What is the structure of acrylamide, bis-acrylamide and polyacrylamide?
4. This protocol describes a discontinuous system, it uses a stacking gel and a running gel. What is the
significance of each layer?
5. What is the role of SDS in SDS-PAGE?
6. What would be the effect on the experiment if:
a. bis-acrylamide was not added to the monomer solution.
b. the gel solution was highly aerated.
c. the APS used was not freshly prepared.
d. there are bubbles in the polymerized gel.
e. too little or too much protein was loaded into the well.
7. What information regarding proteins can be gathered from SDS-PAGE?
8. What are some specific applications for PAGE?

References:
Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. (2014). Molecular Biology of the Cell (6th Edition). USA:
Garland Publishing, Inc.
Chang, M. M., & Lovett, J. (2011). A laboratory exercise illustrating the sensitivity and specificity of Western blot
analysis. Biochemistry and Molecular Biology Education, 39(4), 291-297.
Hoefer. Protein Electrophoresis Applications Guide. 1994. Hoefer Scientific Instruments.
Sambrook J, Green MR. (2014). Molecular Cloning: A Laboratory Manual (4th Edition). USA: Cold Spring Harbor
Press.
Sharma, N., Sharma, R., Rajput, Y. S., Mann, B., Singh, R., & Gandhi, K. (2020). Separation methods for milk
proteins on polyacrylamide gel electrophoresis: Critical analysis and options for better
resolution. International Dairy Journal, 104920.

Appendix: Preparation of Reagents for SDS-PAGE

1. Monomer Solution (40%, 37.5:1)
60 g acrylamide CAUTION: Acrylamide is neurotoxic and should be handled with care
1.6 g bisacrylamide
ddH2O to 200 mL
Store up to 3 months at 4°C in the dark.

2. 4x Running Gel Buffer (1.5 M Tris-Cl, pH 8.8)

36.3 g Tris
Add 150 mL ddH2O
Adjust to pH 8.8 with HCl
ddH2O to 200 mL
Store up to 3 months at 4°C in the dark

3. 4x Stacking Gel Buffer (0.5 M Tris-Cl, pH 6.8)

2.0 g Tris
Add 40 mL ddH2O
Adjust to pH 6.8 with HCl
ddH2O to 50 mL
Store up to 3 months at 4°C in the dark

4. 10% SDS
10 g SDS
ddH2O to 100 mL
Store up to 6 months at room temperature.

5. 10% Ammonium Persulfate (APS)

0.1 g APS
ddH2O to 1.0 mL
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Use fresh, do not store.

6. Water saturated n-Butanol

50 mL n-butanol
5mL ddH2O
Combine in a bottle and shake. Use top phase to overlay gels.
Store at room temperature indefinitely.

7. Running Gel Overlay (0.375 M Tris-Cl, 0.1% SDS, pH 8.8)

25 mL running gel buffer
1.0 mL 10% SDS
ddH2O to 100 mL
Store up to 3 months at 4°C in the dark

8. 4x Treatment Buffer (0.125 M Tris-Cl, 4% SDS, 20% v/v Glycerol, 0.2 M DTT (or 10% β-
mercaptoethanol), 0.02% Bromophenol Blue, pH 6.8)
2.5 ml 4x stacking gel buffer
4.0 mL 10% SDS
2.0 mL glycerol
0.31 g dithiothreitol (or 1 ml β-mercaptoethanol)
2.0 µg bromphenol blue
ddH2O to 10 mL
Store in 0.5 mL aliquots at - 20°C for up to 6 months.

9. Tank Buffer (0.025 M Tris, 0.192 M Glycine, 0.1% SDS, pH 8.3)

30.28 g Tris
144.13 g glycine
10 g SDS
ddH2O to 10 L

10. Staining Solution (0.025% Coomassie Brilliant Blue R 250, 40% methanol, 7% acetic acid)
0.5 g Coomassie Brilliant Blue R
800 mL methanol
Stir until dissolved. Then add:
140 mL acetic acid
ddH2O to 2 L
Store at room temperature for up to 6 months.

11. Destaining solution

700 mL acetic acid
500 mL methanol
ddH2O up to 10 L
Store at room temperature indefinitely.

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