37 Western Blotting Troubleshooting Tips
Western blotting is a technique used to determine the presence or absence of selected proteins in a sample. This online western blotting troubleshooting guide provides solutions for problems such as no bands observed, poor quality transfer and high background. Our guide equips researchers with comprehensive solutions and suggestions to help solve western blotting challenges.
No Bands Observed
| Problem | Explanation |
|
1. Incorrect primary antibody |
Antibody has low to no affinity |
|
2. Inactive antibody |
Perform a dot blot |
|
3. Insufficient protein concentration |
Increase the amount of protein and use a positive control |
|
4. Poor transfer |
Make sure the membrane is activated. Transfer buffer must contain methanol when using nitrocellulose membranes. PVDF membranes must be pre-soaked with methanol |
|
5. Suboptimal transfer time |
High molecular weight proteins may require longer transfer times |
|
6. Incorrect secondary antibody |
Confirm host species and IgG type of primary |
|
7. Antibodies expired |
Check that all antibodies are in date |
|
8. Incorrect storage of antibodies |
Ensure all antibodies are stored as per manufacturer’s instructions |
|
9. Sodium Azide contamination |
Sodium Azide contamination will quench HRP signal |
|
10. Suboptimal primary antibody incubation time |
Increase incubation time with the primary antibody |
|
11. Incompatible primary and secondary antibody |
Maintain a consistent species in both antibodies |
|
12. Insufficient secondary antibody concentration |
Increase the concentration of primary/secondary antibody |
|
13. Excessive washing |
Reduce the number and duration of washes |
|
14. Incorrect orientation |
Mark your membrane to ensure correct orientation |
Poor Quality Transfer
| Problem | Explanation |
|
15. Membrane choice |
Choose either PVDF/nitrocellulose membranes according to the target protein molecular weight |
|
16. Dry membrane |
It is important not to let the membrane or filter paper dry out |
|
17. Incomplete protein resolution |
Ensure optimal gel concentration is used for the protein of interest |
|
18. Incorrect sample preparation |
The sample must contain DTT or B-Mercaptoethanol and be heated prior to loading |
High background
| Problem | Explanation |
|
19. Unspecific antibody binding |
Ensure the correct and most specific primary antibody is used |
|
20. Insufficient blocking |
Optimise blocking time duration |
|
21. Suboptimal antibody concentration |
Optimise antibody concentration |
|
22. Insufficient washing |
Increase the number of washes performed. Increase the concentration of Tween 20 used in wash buffer |
|
23. Incorrect membrane choice |
Nitrocellulose membranes generally have less background compared to PVDF |
|
24. Film overexposed |
Reduce the exposure time |
Too Many Bands
| Problem | Explanation |
|
25. Unspecific antibody |
Ensure the antibody used is specific for the protein of interest |
|
26. Proteolytic breakdown |
Use protease inhibitors to prevent the proteolytic breakdown of the antigen |
|
27. Gel overloading |
Overloading the gel with too much protein can cause the development of “Ghost bands.” Optimise protein concentration. |
|
28. Insufficient blocking |
Extend the blocking time |
|
29. Low antigen concentration |
Consider immunoprecipitating target protein |
|
30. Unspecific secondary antibody binding |
Use secondary antibody only control. If bands develop use a different secondary antibody |
|
31. Analyte aggregation |
Increase DTT concentration |
|
32. Post translational modification |
Protein sample has multiple modified forms e.g. acetylation, methylation and phosphorylation |
|
33. Protein degradation |
Target protein of interest degraded |
|
34. Splice variants |
Could lead to the visualisation of multiple bands |
|
35. High primary antibody concentration |
Use a lower concentration of primary antibody |
Other
| Problem | Explanation |
|
36. Unresolved proteins |
Inefficient separation. Use high molecular weight and low molecular weight proteins |
|
37. Smile/Curve effect on the gel |
Incorrect voltage. Inconsistent temperatures |