Molecular Biology. A Practical Guide - Velikov V.A. 2013
Protein Electrophoresis
Preparation of Solutions and Casting of PAGE
Gel Electrophoresis is one of the standard molecular biology techniques used for analysis. Protein molecules in solution carry a net charge at any pH other than their isoelectric point, which drives their mobility in an electric field. Proteins are typically separated in polyacrylamide gels (PAGE), which feature smaller pore sizes compared to agarose gels. The gel is formed through the polymerization of acrylamide monomer molecules in the presence of $N,N'$-methylenebisacrylamide, which acts as a cross-linking agent. By varying the monomer concentration, gels with custom pore sizes can be produced. PAAG is denser than agarose, resistant to boiling in Water, flexible, and less brittle.
PAGE consists of a stacking gel and a resolving (running) gel. The acrylamide concentration in the resolving gel depends on the protein size: large proteins require gels with lower concentrations starting from 5% and up, whereas smaller proteins are separated in fine-pored gels containing up to 20–25% acrylamide. Gel casting and electrophoretic protein Separation are performed in a vertical orientation.
Numerous modifications of the PAGE Protein Electrophoresis method have been developed to address various analytical challenges and accommodate different PROTEINS AND Peptides. The most widespread variant is denaturing protein electrophoresis in the presence of sodium dodecyl sulfate (a chaotropic agent and surfactant), originally described by Laemmli (1970). One of the standard modifications of this method is outlined below.
Preparing for protein electrophoresis is more time-consuming than DNA electrophoresis. Polyacrylamide gels are typically prepared in advance using stock solutions.
The stacking gel generally has a polyacrylamide concentration of 2% to 8% and a pH of 6.8. The resolving gel features a polyacrylamide concentration ranging from 5% to 20% and a pH of approximately 8.5–8.9. The choice of gel density depends on the molecular weights of the target proteins. Tris-Glycine or Tris-borate buffers with a pH of 8–9 are most commonly used as running buffers.
Materials and Equipment
Vertical electrophoresis apparatus, DC power supply, notched and plain Glass plates, comb, spacers, sample loading syringe, and Reagents for preparing the PAGE and buffers.
Solutions
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1. Electrode buffer (200 mL) Tris-OH (tris(hydroxymethyl)aminomethane) Glycine SDS H2О (bidistilled, deionized, hereinafter) pH 8.4; titrate the glycine solution to the desired pH using dry Tris; add SDS last, after adjusting the pH |
3.03 g 14.44 g 1.0 g to 200 mL |
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2. AA / bisAA solution: AA (acrylamide) bisAA ($N,N'$-methylenebisacrylamide) H2О |
30.0 g 0.8 g to 100 mL |
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3. Buffer 1 (for stacking gel) Tris-HCl (tris-hydrochloride) SDS H2О pH 6.8; titrate with 4–6 M HCl before adding SDS |
6.06 g 0.4 g to 100 mL |
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4. Buffer 2 (lower resolving gel buffer) Tris-OH (tris(hydroxymethyl)aminomethane) SDS H2О pH 8.8; titrate to the required pH with HCl before adding SDS |
18.17 g 0.4 g to 100 mL |
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5. BPB (bromophenol blue) H2О |
2 mg to 4 mL |
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6. Staining solution (0.125% Coomassie R-250) Coomassie Brilliant Blue R-250 (G-250 has lower resolution) Ethanol Acetic acid (10%) H2О |
1.25 mg 5 mL 1 mL to 10 mL |
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7. Washing solution I (50% ethanol, 10% acetic acid) Acetic acid (10%) Ethanol (50%) H2О Dilute 5-fold before use |
10 mL 50 mL to 100 mL |
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8. Washing solution II (5% ethanol, 10% acetic acid) Acetic acid (10%) Ethanol (50%) H2О Dilute 5-fold before use |
35 mL 25 mL to 100 mL |
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2× sample buffer: 15% glycerol |
7.5 mL |
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ß-mercaptoethanol |
2.5 mL |
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SDS |
1.15 g |
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Tris-HCl (tris-hydrochloride) |
0.38 g |
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H2O, pH 6.8 |
to 25 mL |
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- 10% TCA, - 5% APS (ammonium persulfate), - TEMED ($N,N,N',N'$-tetramethylethylenediamine). |
Preparing the Glass Plates for Polyacrylamide Gel Casting
1. Take the notched glass plate and place two spacers along its side edges.
2. Place the glass plate without a notch on top of the spacers. Carefully position the assembled unit vertically in the chamber so that the gel can be poured into the cavity between the glass plates. The unnotched glass plate should face outward toward the researcher. Secure the assembly with clamps.
3. Melt 3% agarose, cool it to 50–60°C, and pour a thin layer (5 mm) onto the bottom of the electrophoresis tank to prevent leakage of the gel solution.
Gel Casting
1. To prepare the resolving gel, mix components a (except for TEMED and APS) according to Table 3 (for various volumes!). Mix the resulting mixture thoroughly b.
Class="center">Table 3. Preparation of the resolving gel
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Component |
3% |
6% |
10% |
20% |
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AA/bisAA solution |
1 mL |
3 mL |
4.95 mL |
13.34 mL |
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Buffer 2 |
2.5 mL |
3.75 mL |
3.75 mL |
5 mL |
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Н2О |
6.5 mL |
8.25 mL |
6.3 mL |
1.66 mL |
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TEMED |
10 µL |
15 µL |
15 µL |
40 µL |
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APS 5% |
30 µL |
45 µL |
45 µL |
120 µL |
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Total V |
10 mL |
15 mL |
15 mL |
20 mL |
2. Add TEMED and APS sequentially and mix the resulting solution thoroughly, taking care to avoid forming gas bubbles during mixing.
3. Gently pour the obtained resolving gel solution between the glass plates so that the level does not exceed 3 cm from the upper edge of the plates.
4. Carefully layer a thin layer of methanol or deionized water onto the resolving gel solution and leave the gel to polymerize (up to 20 min).
5. Prepare the stacking gel solution as indicated in Table 4.
6. Remove the methanol (!) or water layer from The surface of the polymerized resolving gel using filter paper.
7. Layer the stacking gel solution onto the surface of the polymerized resolving gel, and immediately insert the comb between the glass plates.
Table 4. Preparation of the stacking gel
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Component |
6% |
10% |
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AA/bisAA sol. |
2 ml |
3.3 ml |
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Buffer 1 |
2.5 ml |
2.5 ml |
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Н2О |
5.5 ml |
4.2 |
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TEMED |
20 µl |
10 µl |
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APS 5% |
60 µl |
30 µl |
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V total |
10 ml |
10 ml |
8. Leave the gel to set for 20 min c. It is convenient to pour a small amount of the remaining gel solution into a 1.7 ml microcentrifuge tube and use this solution as a polymerization indicator.
Preparing the chamber for electrophoresis
1. Place the electrophoresis chamber on a flat, horizontal surface. Add electrode buffer to the upper chamber reservoir so that the buffer level is 0.5 cm above the top of the gel.
2. Add electrode buffer to the lower buffer tank of the chamber.
3. Carefully remove the comb and wash the wells with electrophoresis buffer using a syringe or micropipette.
Notes
a The ready-made stock solution for the gel at the desired concentration, excluding TEMED and APS, can be stored in the refrigerator for about a year. Acrylamide is toxic; always wear gloves while handling!
b 15% PAGE gels effectively resolve proteins with molecular weights ranging from 10-45 kDa, 10% gels from 14-205 kDa, 7.5% gels from 24-205 kDa, and 5% gels from 36-205 kDa.
c Prepared gels can be stored wrapped in the refrigerator without removing the comb. However, since the resolving and stacking gels use different buffers, it is best to use the freshly poured gel immediately.
Last update: 13/08/2026
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