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Western Blotting Troubleshooting Tips

Western Blotting Troubleshooting Guide

Over 30 great western blotting troubleshooting tips for problems such as no bands observed and high background.

No Bands Observed

1.       Incorrect primary antibody used Antibody used has a low affinity for the protein of interest.
2.       Antibody lost activity Antibody may have lost activity – perform a dot plot
3.       Not enough protein loaded onto gel Increase the amount of protein and use a positive control.
4.       Poor Transfer Make sure the membrane is activated. Either with Methanol for PVDF, for nitrocellulose membranes use transfer buffer to wet the wet and activate the membrane.
5.       Transfer time too long or too short Optimise. High molecular weight may require a longer transfer time in comparison to low molecular weight proteins
6.       Incorrect secondary antibody used Confirm host species and IgG type of primary
7.       Antibodies expired Check all antibodies are not out of date
8.       Incorrect storage of antibodies Ensure all antibodies are stored as per manufacturer’s instructions.
9.       Sodium Azide contamination Sodium Azide contamination will quench HRP signal
10.   Primary antibody incubation time too short Increase incubation time with the primary antibody to overnight
11.   Primary and secondary antibody not compatible The secondary antibody used must be raised against the species the primary antibody was raised in.
12.   Not enough primary secondary antibody used Increase the concentration of primary/secondary antibody used
13.   Excessive washing Reduce the number and duration of washes

Poor Quality Transfer

14.   Poor Quality membrane PVDF membranes are considered to be the best for low molecular weight proteins.
15.   Check transfer protocol Ensure you are following the transfer correctly
16.   Filter Paper Dried out It is important not to let the membrane or filter paper dry out.
17.   SDS PAGE Gel Check the gel was made correctly and the correct percentage used for the protein of interest
18.   Sample preparation Incorrect sample preparation. The sample must contain DTT or B-Mercaptoethanol and be heated prior to loading

High background

19.   Non-specific binding of primary antibody Ensure the correct and most specific primary antibody is used
20.   Insufficient blocking Extend blocking time to overnight
21.   Antibody used too concentrated Decrease the concentration of antibody used
22.   Insufficient Washing Increases the number of washes performed. Increase the concentration of tween 20 used in wash buffer.
23.   Choice of membrane used Nitrocellulose membranes generally have less background compared to PVDF
24.   Film Overexposed Reduce the exposure time

Too many bands

25.   Antibody not specific enough Ensure the antibody used is specific for the protein of interest
26.   Proteolytic breakdown Use protease inhibitors to prevent the proteolytic breakdown of the antigen
27.   Overloading the gel Overloading the gel with too much protein can cause the development of “Ghost bands” try a dilution to optimise the amount of protein to use
28.   Insufficient blocking Extend to blocking time to overnight
29.   Concentration of antigen too low Try Immunoprecipitation
30.   Non- specific binding of the secondary antibody Run a control of the secondary antibody alone. If bands develop use a different secondary antibody
31.   Analyte Aggregation Increase the amount of DTT used to reduce disulphide bonds.
32.   Protein sample has multiple modified forms Eg acetylation, methylation and phosphorylation
33.   Target digested The target in your protein sample has been digested
34.   Splice variants and unreported novel proteins These effect the efficacy of antibody binding
35.   Primary antibody concentration too high Use a lower concentration of primary antibody


36.   Bands of interest too low or high Separation not efficient. Use a higher percentage gel for small proteins and a lower percentage gel for larger proteins.
37.   Smile/Curve effect on bands Migration voltage too fast. Furthermore fill empty well with loading buffer.
38.   Know your orientation Cut out a small section and the top of your membrane to ensure the orientation is always known.