| 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 |
