From appearance to evidence
Western blot diagnostic case library
Each scenario starts from an observation and asks what evidence would distinguish competing causes. These are teaching frameworks, not claims that the website performed the experiments shown.
How to read these cases
Causes are ordered by what should be tested first, not by a calculated probability. Every image is an original teaching illustration that presents evidence relationships; it is not raw experimental data and does not replace positive or negative controls.
Case 1
Blank blot with little protein on the membrane
Observation: Target, loading control and positive control are absent. The transfer stain shows little or no protein across all lanes.
Blank-transfer evidence comparison
Original teaching illustration placing the blot appearance and the evidence to inspect in one view.
Evidence to collect
- • Uncropped membrane image
- • Post-transfer gel or total-protein stain
- • Sandwich orientation and electrode record
- • Transfer current/voltage trace
Causes to test first
- 1. Reversed sandwich or polarity
- 2. Poor electrical contact or exhausted buffer
- 3. Air gap affecting most of the membrane
Next discriminating action
Repeat transfer with the antibody and detection conditions unchanged; document sandwich orientation and total-protein stain.
What the current appearance cannot prove
A blank antibody image alone cannot distinguish failed transfer from failed detection.
Case 2
Loading control present but a large target is weak
Observation: A routine loading control is clear, but a high-molecular-weight target is absent or much weaker than expected.
Large-target under-transfer evidence
Original teaching illustration placing the blot appearance and the evidence to inspect in one view.
Evidence to collect
- • Validated positive sample
- • Target-size region on the uncropped membrane
- • Transfer stain at high molecular weight
- • Post-transfer gel if available
Causes to test first
- 1. Under-transfer of the large target
- 2. Poor extraction of the relevant compartment
- 3. Low biological abundance or wrong collection time
Next discriminating action
Run a validated positive control and inspect transfer evidence before changing antibody concentration.
What the current appearance cannot prove
A small loading-control band does not prove that a much larger target transferred efficiently.
Case 3
Small target disappears despite acceptable general transfer
Observation: Most proteins transfer, but the expected low-molecular-weight target is absent or inconsistent.
Small-protein loss during transfer
Original teaching illustration placing the blot appearance and the evidence to inspect in one view.
Evidence to collect
- • Membrane pore size
- • Transfer duration and methanol content
- • Post-transfer gel
- • A validated small-protein positive control
Causes to test first
- 1. Blow-through or poor retention
- 2. Target below the cropped region
- 3. Antibody incompatibility or low abundance
Next discriminating action
Compare a shorter transfer or 0.2 µm membrane while keeping antibody conditions fixed.
What the current appearance cannot prove
The absence of a low band does not by itself prove blow-through.
Case 4
Uniform dark background across the membrane
Observation: The whole membrane is gray or dark and useful bands appear only at very short exposure.
Uniform-background exposure comparison
Original teaching illustration placing the blot appearance and the evidence to inspect in one view.
Evidence to collect
- • Exposure series
- • Secondary dilution and species
- • Substrate preparation record
- • Wash volume and duration
Causes to test first
- 1. Excess secondary antibody
- 2. Overexposure or contaminated substrate
- 3. Insufficient washing or membrane drying
Next discriminating action
Keep the primary condition fixed and change either secondary concentration or exposure first.
What the current appearance cannot prove
The visual pattern alone cannot prove that blocking is the main cause.
Case 5
Reproducible extra bands at unexpected molecular weights
Observation: The expected band is present, but one or more additional bands recur across experiments.
Extra-band control comparison
Original teaching illustration placing the blot appearance and the evidence to inspect in one view.
Evidence to collect
- • Knockout/knockdown or known-negative sample
- • Known isoforms and processing
- • Independent antibody against another epitope
- • Sample degradation record
Causes to test first
- 1. True isoform, cleavage or post-translational modification
- 2. Primary-antibody cross-reactivity
- 3. Degradation or endogenous immunoglobulin signal
Next discriminating action
Use a genetic negative control or orthogonal antibody before labeling the extra band nonspecific.
What the current appearance cannot prove
Molecular weight alone cannot establish whether a band is biological or technical.
Case 6
Vertical smear and broad lanes after high loading
Observation: Lane boundaries are poor and signal spreads vertically, especially in the most concentrated samples.
Loading-dependent smear progression
Original teaching illustration placing the blot appearance and the evidence to inspect in one view.
Evidence to collect
- • Protein load series
- • Lysate viscosity and clarification notes
- • Salt/detergent composition
- • Freeze–thaw history
Causes to test first
- 1. Sample overload
- 2. Viscous or insufficiently clarified lysate
- 3. Degradation or gel incompatibility
Next discriminating action
Keep antibody conditions unchanged and run a lower loading series first.
What the current appearance cannot prove
A smear can also be real for heavily modified proteins and must be checked biologically.
Case 7
Local blank area consistent with a transfer bubble
Observation: A sharply bounded pale region appears at a fixed membrane position and cuts across adjacent lanes.
Transfer-bubble position match
Original teaching illustration placing the blot appearance and the evidence to inspect in one view.
Evidence to collect
- • Ponceau or total-protein image
- • Transfer sandwich photo or assembly record
- • Artifact shape across targets
- • Membrane and filter-paper inspection
Causes to test first
- 1. Bubble between gel and membrane
- 2. Fold or damaged filter paper
- 3. Uneven cassette pressure
Next discriminating action
Reassemble with deliberate bubble removal and verify the whole membrane before antibody incubation.
What the current appearance cannot prove
Position-dependent loss should not be interpreted as sample-specific biology.
Case 8
Loading control is saturated and distorts normalization
Observation: The loading-control bands look equally black across lanes, while target differences appear large.
Saturation and normalization bias
Original teaching illustration placing the blot appearance and the evidence to inspect in one view.
Evidence to collect
- • Shorter exposure series
- • Raw pixel saturation check
- • Protein loading dilution series
- • Total-protein normalization image
Causes to test first
- 1. Loading control outside the linear range
- 2. Target and control acquired under incompatible exposure conditions
- 3. Unstable loading control under treatment
Next discriminating action
Repeat acquisition or loading so both target and normalization signals are inside a demonstrated linear range.
What the current appearance cannot prove
A visually uniform black control is not evidence of equal loading when saturated.