Low or No Signal on Western Blot (Western Blot (CST Guide))

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Target protein bands are absent or very faint on the blot, even after extended exposure times. Positive control samples may also show weak or no signal. Common causes: 1) Reusing pre-diluted antibodies: diluted antibody is less stable and prone to microbial contamination 2) Insufficient protein loading: less than 20-30 µg per lane for whole cell extracts, or less than 100 µg per lane for modified targets in tissue extracts 3) Low basal expression of phosphorylated or modified proteins without appropriate treatment or stimulation 4) Incomplete cell lysis without sonication: membrane-bound and organelle proteins poorly extracted, nuclear DNA interferes with gel loading 5) Wrong primary antibody dilution buffer: non-fat dry milk too stringent for many antibodies, reducing sensitivity 6) Sub-optimal transfer conditions: standard 20% methanol transfer buffer causes poor transfer of high MW proteins (>100 kDa) Solutions: 1) Always use freshly diluted antibody for each experiment; discard diluted antibody after use 2) Load at least 20-30 µg protein per lane for whole cell extracts; increase to 100 µg per lane for modified targets in tissue extracts 3) Include protease inhibitors: leupeptin (1.0 µg/ml), PMSF, and phosphatase inhibitors: sodium pyrophosphate (2.5 mM), beta-glycerophosphate (1.0 mM), sodium orthovanadate (2.5 mM) 4) Perform sonication: 3 × 10 second bursts with microtip probe sonicator at 15W (50% power) on ice for 1 ml samples 5) Use recommended antibody dilution buffer from product datasheet (5% BSA or 5% non-fat dry milk in 1X TBS/0.1% Tween-20) 6) For high MW proteins: reduce methanol to 5-10% in transfer buffer and increase transfer time to 3-4 hours at 200-250 mA