Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.
Entire membrane shows elevated signal intensity, making it difficult to distinguish specific protein bands from background noise. Signal-to-noise ratio is poor.
Film shows 'bleached' or 'burnt-out' white bands against dark background instead of expected dark bands. Bands appear as negative images where high protein concentration exists.
Proteins below 15-20 kDa show weak or absent signal while larger proteins are detected normally. Small protein bands appear diffuse or masked.
Replicate Western blots show high variability in band intensity measurements. Coefficient of variation exceeds acceptable limits (>15-20%) making quantitative comparisons unreliable.
Expected protein bands appear faint or barely visible on film or imaging system, even after extended exposure times.
No protein bands are visible on the blot, even at the expected molecular weight position. Film or imaging system shows no detectable chemiluminescence or fluorescence.
Membrane shows irregular signal distribution with visible fingerprints, fold marks, or forceps imprints. Signal intensity varies across membrane surface in non-biological pattern.
Membrane shows scattered dark spots or speckles across the surface, distinct from specific protein bands. Background appears grainy or particulate rather than uniformly elevated.
Blot shows unexpected extra bands at different molecular weights than predicted, or expected band appears at wrong size. Multiple bands may appear where single band is anticipated.
Quantitative measurements show high variability between replicates. Large error bars or standard deviations prevent statistical significance despite apparent differences.
Membrane shows fingerprints, fold marks, or forceps imprints creating irregular patterns. Uneven signal distribution not related to protein bands.
Film shows negative or reverse image with white or 'bleached' bands on dark background instead of expected dark bands. Also called 'burnt-out' bands.
Unexpected bands appear at molecular weights different from target protein. Multiple bands present instead of single expected band, or band appears at incorrect size.
When using rapid immunodetection methods, membrane shows elevated background specific to fast protocols. Issue not present with standard overnight protocols.
Overall high background across entire membrane reduces signal-to-noise ratio. Specific bands difficult to distinguish from background noise.
No bands are visible on the Western blot membrane or film. Complete absence of signal despite confirmed protein loading and transfer.
Proteins smaller than 15-20 kDa show weak or absent signal while larger proteins detect normally. Small protein bands are masked or obscured.
Protein bands appear faint or barely visible on the Western blot membrane or film despite proper loading. Signal intensity is insufficient for detection or quantification.
Discrete spots or speckles appear across the membrane background rather than uniform signal. Pattern resembles aggregates or particulates.
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