Problem Possible Cause Solution
No Signal Inactive enzyme on secondary antibodyTest 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 targetAvoid antibody freeze-thaw cycles; use a fresh aliquot of antibody. Make sure correct antibodies were used and not expired.
 No transfer of target to membraneUse 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 membraneEnsure correct orientation of the blot when adding detection reagents and during exposure.
 Protein degradation due to storagePrepare new blot.
  Inefficient transfer to membraneUse 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.
Weak signalLow antibody concentrationOptimize dilutions for each antibody.
 Antigen washed away by excessive washingReduce number and/or length of washes.
 Reduced activity of substrateEnsure substrate does not have microbial or enzyme contamination. Use fresh aliquot of substrate.
 UnderexposureIncrease exposure time.
  Inadequate blockingIncrease blocking concentration or change blocking buffer.
 Insufficient washesIncrease number and/or length of washes.
 Excess substrateRemove excess substrate by blotting on filter paper prior to exposure.
 Overexposure Decrease exposure time.
High BackgroundExcessive secondary antibodyReduce amount of secondary antibody diluted in blocking buffer.
 Poor quality antibodiesUse high quality purified antibodies; do not use antibodies if expired or have gone through multiple freeze-thaw cycles.
 Contaminated reagents or equipmentUse 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 BackgroundAggregate formation in conjugate antibodyFilter conjugate through 0.2μm filter or use fresh conjugate.
 Areas of membrane dried during incubation stepsDo not allow membranes to dry during any incubation steps. Use agitation during all incubation steps.
 Improper handling of membranesAlways use clean gloves and forceps when handling membranes.
 Contaminated buffers Use new, filtered buffers.
White (Ghost) BandsHigh protein concentrationDilute sample and run again.
 Primary or secondary antibody too concentratedOptimize primary and secondary antibody dilutions.

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