WESTERN BLOTTING: TROUBLESHOOTING GUIDE
Gels
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Smeared gels may be the result of sample overloading or due to high concentrations of DNA.
Partial protein reduction prior to electrophoresis may result in anomalous protein migration in SDSPAGE. We recommend the use of DTT over 2-mercaptoethanol in the sample buffer as it is a more
powerful reducing agent.
Protein Transfer
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Protein binding can be blocked by oils or other proteins. Wear gloves and use fresh nitrocellulose sheets.
The membrane must dry completely before processing the nitrocellulose by blocking or staining. This
will reduce the possibility of transferred protein loss during subsequent steps. Treating with isopropanol
for 1 min. is an alternative to drying.
Small molecular weight proteins may transfer through the membrane. Shorten transfer time, using a
double layer of membrane, or use a PVDF membrane to avoid this problem.
High molecular weight proteins are sometimes more difficult to extract from the gel. Avoid using
methanol by switching to a nylon membrane or use a higher field strength or longer transfer time to avoid
this problem.
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Background Staining
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Diffuse backgrounds may be caused by dirty gel plates, contaminated solutions, membrane spoilage,
insufficient blocking time, incompatible blocking buffer, or excess antibody concentration.
Multiple low molecular weight bands may be due to sample degradation caused by proteases. Add sample
buffer including protease inhibitors immediately after harvesting the sample in order to prevent sample
degradation .
Secondary antibodies sometimes generate extra bands. Run a control experiment by probing the
membrane with the secondary antibody only (i.e. omitting the primary antibody incubation step) to
determine if this is occurring
Additional bands may be due to extended incubation times, sample overloading, or excess exposure times
when developing. Raising the incubation temperature (e.g. to 37C) often works better than lengthening
the incubation time.
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Weak or No Signal
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Load more protein on the gel if constitutive levels of the protein of interest are low.
Detergents may disrupt protein binding. A mild detergent such as Tween-20 is recommended over SDS,
Nonidet P-40, and Triton X-100 for washing and buffers.
For maximimum signal use PVDF membranes. However, PDF membranes may require more stringent
blocking than nitrocellulose.
The secondary antibody conjugate may be inhibited by reagents in the blocking agent or incubation
solution. Horseradish peroxidase should not be used in conjunction with sodium azide or haemoglobin.
Biotinlyated antibodies should not be used in conjunction with milk or casein.
Subjecting the sample to repeated freeze/thaw cycles will degrade the antigen. It is recommended that
large samples be aliquotted to smaller, useable portions for long term storage.
ECL detection is more sensitive than colorimetric.
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