Troubleshooting Guide for SDS-PAGE Protein Electrophoresis |
| Problem |
Possible causes |
Solution |
| Faint or missing protein bands |
Load quantity is below the detection level of the stain |
Check the A280 and increase sample concentration |
| |
|
Use a more sensitive stain (e.g. silver stain) |
| |
Proteins were not fixed in the gel |
Use a stain which also fixes the proteins |
| |
|
Use gel fixing solution |
| |
Small peptides (<4 kDa) did not fix in the gel |
Fix the gel with 5% glutaraldehyde. Rinse the gel well with water before staining |
| |
Proteins are degraded |
Check the A280 and avoid protease contamination |
| |
Protein ran off the gel |
Use a higher concentration PAGE gel. See precast gels for recommended gel concentration or use a 4-20% gel if the size is unknown |
| Film on gel after staining |
Precipitated Coomassie Blue R |
Rinse the gel for 15 seconds in methanol and immediately return to water or destain |
| Poor band resolution |
Concentration of protein is high |
Load 10 μg per protein or 100 μg per protein extract |
| |
Age of the gel, due to base catalyzed |
Order fresh precast gels or cast a fresh gel |
| |
Improper gel concentration |
See precast gels for recommended gel concentration or use a 4-20% gel if the size is unknown |
| Band smearing |
High salt concentrations |
Dialyze sample, precipitate the protein with TCA or use desalting columns |
| |
Concentration of protein is high |
Load 10 μg per protein or 100 μg per protein extract |
| |
Protein aggregation |
Add 4-8 M urea to the sample |
| |
Voltage is high |
Electrophorese at 10-15 V/cm |
| Protein precipitation in the well |
Hydrophobic proteins |
Add 4-8 M urea to the sample |
| White precipitate in sample |
SDS precipitation |
Could be due to the presence of guanidine or potassium salts in the sample |
