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