Possible cause | Solution |
Antibodies may have lost activity | Test antibodies by performing a dot blot |
Insufficient amount of antigen present | Load more protein on gel. If the specific antigen concentration is too low, one can enhance the antigen by immunoprecipitation or fractionation |
Weak antigen expression | Remove Tween during primary antibody incubation to improve antibody binding |
Antibody exposure time is too short | Increase the exposure time |
Insufficient antigen binding to membrane | Use instead a membrane with a smaller pore size or a different type of membrane |
Substrate has lost activity | Test the substrate using a positive control to ensure activity |
Antigen is masked by the blocking buffer | Experiment with other blocking buffers and concentrations such as BSA in Tris-buffer saline, serum, milk, and phosphate-buffered saline |
Air bubbles between gel and membrane | Remove air bubbles by gently rolling over the membrane sandwich with a pipette |
Insufficient transfer time or current | Increase transfer duration for large molecular weight proteins |
Detection enzyme may be inactivated | Avoid sodium azide in HRP-labeled reagents and ensure HRP conjugates are bacteria-free |
Excessive washing of the membrane | Reduce the number of washes to prevent loss of signal |
Excess methanol in the transfer buffer | Find the right balance as too much methanol decreases protein transfer efficiency |