Western Immunoblotting Troubleshooting Guide

Problem: High Background

General background is high or nonspecific bands appear after a 1–30 second exposure of blot to film.

Cause:	Solution:
Primary antibody was not incubated at the correct dilution or was in the incorrect buffer.	Incubate primary antibody overnight at 4°C in TBS/T at the recommended dilution with the appropriate blocking agent. Polyclonal and rabbit monoclonal antibodies give the highest signal/noise in 5% BSA. Mouse monoclonal antibodies give the highest signal/noise in 5% milk. For individual antibodies please consult their respective datasheets for recommended dilution buffer.
Membrane is inappropriate for chemiluminescent detection.	Use only high quality nitrocellulose or PVDF membranes. Select a membrane found to exhibit high specific binding with low background. None of the nylon membranes tested were adequate for Western blotting.
Membrane blocking is not sufficient to prevent nonspecific binding of primary or secondary antibody.	Block for 1 hour at room temperature in 5% milk in TBS/T. Do not block excessively.
Secondary antibody was poor, incubated at the incorrect dilution, or in the incorrect buffer.	Some secondary antibodies bind nonspecifically to proteins in cell extracts. To assess the quality of a secondary antibody, perform a blot (through to film exposure) without primary antibody to determine whether this is a problem. Also, perform serial dilutions of the secondary antibody on blots with the same cell extracts and primary antibody to optimize concentration. Always incubate the secondary antibody in TBS/T, 5% milk for 1 hour in room temperature.
LumiGLO® reagent was prepared using poor quality water.	Chemiluminescent detection requires that high purity water be used to dilute LumiGLO® reagents A and B. Use only Milli-Q or other high quality water with organic and inorganic impurities removed.

Problem: Low Signal

The protein of interest cannot be detected after 1–30 second exposure of blot to film.

Cause:	Solution:
Insufficient incubation with primary antibody.	Phospho-antibodies generated against a single or dual phosphorylation site are highly specific but generally result in lower signal than regular antibodies. It is critical that these antibodies be incubated with blots overnight at 4°C in the appropriate buffer.
Incomplete transfer of proteins to membrane.	Transfer longer or at higher voltage. Monitor transfer using prestained molecular weight markers (membrane staining can inhibit chemiluminescent detection).
Blocking was excessive.	Blocking the membrane for too long in milk can obscure antigenic epitopes and strip proteins from the blot. Block for only 1 hour (never overnight).
Protein of interest is below detectable levels.	20 µg total protein per lane is usually sufficient; more protein can be loaded if constitutive levels of the protein of interest are low. Load more sample or immunoprecipitate before SDS-PAGE. Try other cell lines or tissue sources where protein is more abundant.
Secondary antibody was weak.	Replace secondary antibody or increase the concentration in that step.
LumiGLO® reagent was improperly prepared or stock reagents A and B have become cross-contaminated.	Remake LumiGLO® reagent. Biotinylated molecular weight standards detected with anti-biotin-HRP act as positive controls for chemiluminescent detection.