Molecular Biology: A Practical Guide - Velikov V.A. 2013
Protein Electrophoresis
Performing Protein Electrophoresis
A protein molecule in solution possesses a certain average net integer charge (either positive or negative) at any pH value other than its isoelectric point. This drives the migration of molecules through a polyacrylamide gel under METABOLISM/18.html">The Influence of an external electric field. In addition to charge, protein mobility is also affected by molecular size.
Electrophoresis is typically performed at neutral or slightly alkaline pH values, where most Proteins migrate toward the anode, similarly to DNA in electrophoresis. The progress of electrophoresis is monitored by the movement of the bromophenol blue (BPB) tracking dye. The dye migrates with the electrophoresis front, and no proteins move ahead of it.
Materials and Equipment
Electrophoresis gel chamber, power supply, protein samples for analysis, and molecular weight markers.
Solutions
- 2× sample loading buffer (Practical 11.1).
- BPB solution (Practical 11.1).
1. Prepare the samples so that the total protein concentration does not exceed 10 mg/ml. Dilute 10 µl of the sample 1:1 with 2× sample loading buffer and add 4 µl of the BPB solution. It is recommended to boil the samples for 5 min.
2. Using a Hamilton syringe or a micropipette with an extended tip, load 10 µl of each sample into the gel wells.
3. Connect the electrophoresis chamber to the power supply, ensuring correct polarity.
4. Perform electrophoretic Separation of proteins at a constant current of 20 mA, 60 V for a single gel, and 60 mA, 150 V for another gel. The voltage should be increased once the BPB front reaches the resolving (lower) gel. Under these conditions, electrophoresis takes 40–50 min.
5. Once the dye reaches the bottom edge of the gel, turn off the power.
6. Remove the gel cassette from the electrophoresis chamber.
7. Carefully remove the spacers located between the Glass plates.
8. Separate the gel from the glass plates a and proceed with staining.
Note
a To impart hydrophobic properties to the glass plates for easier gel detachment, they are often pre-silanized using bind-silane and repel-silane.
Last update: 13/08/2026
Editorial and Educational Adaptation: This material has been compiled based on the primary/original source text. The project team performed an editorial review, corrected technical inaccuracies, structured sections, and adapted the content for an educational format.
What was processed:
- elimination of formatting defects (OCR errors, structural breaks, corrupted characters);
- editorial organization of content;
- standardization of terminology in accordance with academic sources;
- verification of factual statements against the original source text.
All mentions of the author, publication year, and origin of the primary text have been preserved in accordance with the source.