Western Blot FAQ Analysis

Problem	Possible causes	Solution
High background value	The membrane is not uniformly infiltrated	PVDF membrane Before membrane transfer, completely soak the membrane in 100% methanol, and soak it in buffer for 10 minutes before membrane transfer
High background value	Membrane or buffer contamination	Wear gloves when taking the membrane and blotting paper, and replace with fresh transfer buffer
High background value	Insufficient blocking	Replace the blocking solution or extend the blocking time
High background value	Antibody cross-reacts with blocking solution	Determine whether there is cross-reactivity between the primary antibody, secondary antibody and blocking solution
High background value	Antibody concentration is too high	Do a pre-experiment to determine the optimal working concentration of the primary antibody and secondary antibody
With a lot of noise	Primary antibody is not unique	Prepare monoclonal antibodies or reselect the site of the synthetic antigen peptide to prepare antibodies
With a lot of noise	Secondary antibody binds non-specifically	Set up a parallel control without adding primary antibody and only adding secondary antibody to detect whether the secondary antibody binds non-specifically
With a lot of noise	Different splice variants	Select the region specific to the antigen for immunization
No signal or weak signal	Antibody is not suitable for WB	Set up positive controls and internal reference proteins
No signal or weak signal	Antibody concentration is too low	Increase the concentration of the antibody
No signal or weak signal	Antibody is not stored properly	Divide and store to avoid repeated freezing and thawing
No signal or weak signal	Insufficient antigen	Increase the amount of protein loaded
No signal or weak signal	Membrane is over-rinsed	Reduce the time and number of rinses
No signal or weak signal	Transfer is not sufficient	Extend the membrane transfer time or increase the membrane transfer voltage

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