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.). |
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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. |