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 usedAntibody used has a low affinity for the protein of interest.
2.       Antibody lost activityAntibody may have lost activity – perform a dot plot
3.       Not enough protein loaded onto gelIncrease the amount of protein and use a positive control.
4.       Poor TransferMake 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 shortOptimise. High molecular weight may require a longer transfer time in comparison to low molecular weight proteins <10kDa which require a lower voltage.
6.       Incorrect secondary antibody usedConfirm host species and IgG type of primary
7.       Antibodies expiredCheck all antibodies are not out of date
8.       Incorrect storage of antibodiesEnsure all antibodies are stored as per manufacturer’s instructions.
9.       Sodium Azide contaminationSodium Azide contamination will quench HRP signal
10.   Primary antibody incubation time too shortIncrease incubation time with the primary antibody to overnight
11.   Primary and secondary antibody not compatibleThe secondary antibody used must be raised against the species the primary antibody was raised in.
12.   Not enough primary secondary antibody usedIncrease the concentration of primary/secondary antibody used
13.   Excessive washingReduce the number and duration of washes

Poor Quality Transfer

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

High background

19.   Non-specific binding of primary antibodyEnsure the correct and most specific primary antibody is used
20.   Insufficient blockingExtend blocking time to overnight
21.   Antibody used too concentratedDecrease the concentration of antibody used
22.   Insufficient WashingIncreases the number of washes performed. Increase the concentration of tween 20 used in wash buffer.
23.   Choice of membrane usedNitrocellulose membranes generally have less background compared to PVDF
24.   Film OverexposedReduce the exposure time

Too many bands

25.   Antibody not specific enoughEnsure the antibody used is specific for the protein of interest
26.   Proteolytic breakdownUse protease inhibitors to prevent the proteolytic breakdown of the antigen
27.   Overloading the gelOverloading 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 blockingExtend to blocking time to overnight
29.   Concentration of antigen too lowTry Immunoprecipitation
30.   Non- specific binding of the secondary antibodyRun a control of the secondary antibody alone. If bands develop use a different secondary antibody
31.   Analyte AggregationIncrease the amount of DTT used to reduce disulphide bonds.
32.   Protein sample has multiple modified formsEg acetylation, methylation and phosphorylation
33.   Target digestedThe target in your protein sample has been digested
34.   Splice variants and unreported novel proteinsThese effect the efficacy of antibody binding
35.   Primary antibody concentration too highUse a lower concentration of primary antibody


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