The Western Blot technique for identification of specific proteins may have a number of problems that need to be resolved. One of these is an absent or weak signal.
Situation: no or weak signal
Problem
Possible Solution
inefficient or incomplete transfer
see below
insufficient binding to the membrane
Change membrane to smaller pore size. Change membrane type. If antigen has HMW add SDS to transfer buffer. If antigen has LMW, add 20% methanol to transfer buffer and reduce transfer time.
antibody concentration too low
Increase antibody concentration. If loss of antibody activity suspected, perform dot blot to determine activity.
antibody has poor affinity for target protein
Use antibody with high affinity for the target protein.
insufficient antigen present
Load more protein onto the gel.
antigen masked by blocking buffer
Decrease concentration of protein in blocking buffer. Try a different blocker buffer.
Increase incubation time of membrane with substrate. Increase film exposure time. Ensure that the substrate is not expired. Use a maximum sensitivity substrate to maximize signals.
reprobing after membrane stripped
Avoid repeated stripping of the same membrane. Shorten incubation in stripping buffer to prevent loss of antigen.
antigen digestion on membrane
Proteolytic activity in blocking substance.
protein degradation (prolonged storage, improper storage, etc)
Prepare new blot. Correct any storage problems.
Possible solutions for inefficient/incomplete transfer:
(1) Increase transfer time.
(2) Increase voltage.
(3) Make sure that conditions of sample preparation have not destroyed antigen.
(4) Use molecular weight markers compatible with a western-imaging substrate.
(5) Wet and activate the membrane according to the manufacturer's instructions.
(6) Ensure that the stack is placed in the transfer apparatus in the proper orientation.
(7) Ensure sufficient contact between the gel and membrane during transfer, using a gel roller across the transfer stack.
(8) Stain the gel after transfer with a total protein stain to determine transfer efficiency.
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