posted this at Jun 5, 2025 Updated at Oct 29, 2024
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
For more product details, please visit Aladdin Scientific website.
