Western Blot Troubleshooting Guide
Diffuse bands
Possible Cause
1. Antibody concentration too high
1. Decrease the antibody concentration
2. Excess protein on gel
2. Reduce the amount of total protein loaded on the gel
3. Protein transfer was too fast and/or the gel
was over-heated during electrophoresis
3. Increase transfer time
Apply cooling system for electrophoresis
1. Wash blots after transfer and don’t use SDS
Nonspecific bands 1. Non-specific binding to immobilized
protein bands caused by SDS
2. Gradually accumulative differences of
protein expression profiles due to frequent
passage of cell lines
2. Go back to original non-passaged cell line and then
run current and original cell line samples in parallel
3. Degraded protein sample
3. Use fresh sample and add protease
4. Presence of new proteins or different
splice variants that share similar epitopes
4. Check literature for other reports
Perform a BLAST search
Use cell line or tissue reported on the datasheet
5. Use monoclonal antibodies
Purify the antibody by affinity method
5. Presence of impurities in the antibody
6. Formation of multimers on protein target
6. Before running SDS PAGE gel, boil protein for 10 minutes
to disrupt multimers
7. Formation of different protein
subtypes which have different
molecular weights
8. Presence of multi-modifier locus in
7. Examine literature and use bioinformatics analysis to
estimate correct size of protein
9. Too high primary antibody
10. Non-specific bands caused by
secondary antibody
11. Excess protein on gel
9. Decrease primary antibody concentration
12. Insufficient washing
12. Increase number of washes
13. Blocking problem
13. Increase blocking time
Optimize choice of blocking agent
8. Examine literature and use bioinformatics analysis to
confirm presence of modifier locus and its molecular weight
10. Use fluorescence labels on primary antibody
11. Reduce amount of total protein loaded on gel
Weak or no signal from
the blot
Possible Cause
1. Detection step missed or detection
reagents not working
2. Insufficient incubation with
detection reagent
3. Poor or incomplete transfer
4. Use positive control and/or molecular weight marker to
match gel separation range to size of protein being blotted.
After blotting, stain membrane to measure transfer efficiency.
5. Incorrect reagents added or
incorrect containers are filled
5. Make sure that
a. primary and secondary antibodies are added to correct
b. the numbers on the antibody container in the tank and tray
match each other
c. primary and secondary antibodies, substrates, enzyme
system, and sample are compatible
6. Sample is too dilute
6. Load a larger amount of protein onto the gel or increase the
concentration of proteins
7. Use membranes with appropriate binding capacity
Dry the PVDF membrane after protein transfer to ensure
strong binding of the proteins
8. Inactive or overly dilute primary or
secondary antibody
1. Film overexposed or became wet
during exposure
2. Suboptimal blocking time or
washing intensity
3. High concentration of primary
and/or secondary antibody
4. Protein is overloaded
5. Membrane, solutions, trays, or
antibody containers are contaminated.
Non-specific binding
too high
1. After the blot processing is complete, perform the detection
step using your standard detection reagents and protocol
manually. Make sure the detection reagents are functional
2. Remove blot from detection reagent when signal-to-noise
ratio is acceptable
3. Make sure transfer apparatus and membrane sandwiches are
assembled correctly
Use appropriate transfer times
After blotting, stain membrane to measure transfer efficiency
4. Protein of interest ran off the gel
7. Poor retention of proteins or the
protein is weakly bound to membrane
High background on the
8. Determine antibody activity by performing a serial dilution
using six trays or by using dot blot
Increase antibody concentration if necessary
1. Decrease exposure time or allow signal to further decay
Prevent leakage of solutions by encasing membrane in
transparency film and blotting excess substrate from edge
before the exposure
2. Increase the blocking time and the number of washes
3. Determine optimal antibody concentration by performing
dilution series using all six trays of the BlotCycler™.
Decrease antibody concentration as necessary
4. Reduce the amount of protein loaded on the gel or
dilute the sample
5. Use clean glassware and purified water to prepare solutions
Wear clean gloves at all times
Use the forceps when handling membranes
Run the cleaning protocol using the recommended cleaning
buffer while increasing the concentration twofold
1. Insufficient removal of SDS or
weakly bound proteins on
the membrane after blotting
1. Follow proper protocol for membrane preparation before the
immunodetection step
2. The blocking time is too short
2. Increase the blocking time
3. Affinity of the primary antibody for
the protein standards
3. Check with protein standard manufacturer for homologies
with primary antibody
4. Protein is overloaded
4. Reduce the amount of protein loaded on the gel or
dilute the sample
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