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Troubleshooting Western Blot

 

Problem Possible Cause Solution
No Signal Inactive enzyme on secondary antibody Test enzyme activity by luminometer or dotting secondary antibody directly to membrane. AP enzyme should never be frozen; avoid HRP freeze-thaw cycles.
Primary or secondary antibody no longer bind to target Avoid antibody freeze-thaw cycles; use a fresh aliquot of antibody. Make sure correct antibodies were used and not expired.
No transfer of target to membrane Use protein stain to detect proteins on membrane. Verify compatibility of the stain with membrane prior to use.
Contaminated substrate Ensure substrate does not have microbial or enzyme contamination. Use fresh aliquot of substrate.
Substrate added to back of membrane Ensure correct orientation of the blot when adding detection reagents and during exposure.
Protein degradation due to storage Prepare new blot.
Weak signal Inefficient transfer to membrane Use protein stain to detect proteins on membrane. Verify compatibility of the stain with membrane prior to use.
Low protein concentration Load more protein or try a more sensitive substrate.
Incorrect blocking reagent Change blocking reagent or reduce concentration.
Low antibody concentration Optimize dilutions for each antibody.
Antigen washed away by excessive washing Reduce number and/or length of washes.
Reduced activity of substrate Ensure substrate does not have microbial or enzyme contamination. Use fresh aliquot of substrate.
Underexposure Increase exposure time.
High Background Inadequate blocking Increase blocking concentration or change blocking buffer.
Insufficient washes Increase number and/or length of washes.
Excess substrate Remove excess substrate by blotting on filter paper prior to exposure.
Overexposure Decrease exposure time.
Excessive secondary antibody Reduce amount of secondary antibody diluted in blocking buffer.
Poor quality antibodies Use high quality purified antibodies; do not use antibodies if expired or have gone through multiple freeze-thaw cycles.
Contaminated reagents or equipment Use filtered solutions and ensure all equipment is clean
(e.g., containers, forceps, gloves, etc.).
Tween omitted from buffers Include 0.05%-0.1% Tween-20 in blocking and wash buffers.
Blotchy or Speckled Background Aggregate formation in conjugate antibody Filter conjugate through 0.2μm filter or use fresh conjugate.
Areas of membrane dried during incubation steps Do not allow membranes to dry during any incubation steps. Use agitation during all incubation steps.
Improper handling of membranes Always use clean gloves and forceps when handling membranes.
Contaminated buffers Use new, filtered buffers.
White (Ghost) Bands High protein concentration Dilute sample and run again.
Primary or secondary antibody too concentrated Optimize primary and secondary antibody dilutions.