|BioLegend Western Blot Troubleshooting Guide
|What's the issue?
|No or low Signal
|The primary and secondary antibodies are not compatible.
|Ensure you are using secondary antibody that binds to your primary antibody (i.e. if your primary is rat, be sure you are using an anti rat secondary).
|Insufficient primary or secondary antibody has bound to the protein of interest.
|Utilize a higher concentration of antibody and or incubate for a longer period (i.e. overnight at 4°C).
|There is not enough antigen.
|Load a larger amount of protein onto the gel. Use protease inhibitors and run the recommended positive control.
|Overuse of primary antibody.
|Use fresh antibody (the effective concentration is lowered after each use).
|Incubation with detection reagent not sufficient.
|Increase the blots incubation time with detection reagent.
|Detection reagents are not working.
|Make sure detection reagents are functional by testing with a different primary antibody.
|Poor transfer during blotting.
|Make sure the transfer apparatus is set up correctly. Ensure you are using the correct transfer times.
|Secondary antibody is inhibited by sodium azide.
|Do not use sodium azide with HRP-Conjugated antibodies.
|Excessive membrane washing.
|Reduce washing step repetitions or duration.
|Target protein ran off the gel.
|Use a positive control and a molecular weight marker matched to the size range of the target protein.
|The target protein is not found in high concentrations in your sample.
|Maximize the target's concentration by enriching the sample beforehand.
|The primary or secondary antibody binds to the blocking agent.
|Utilize a mild detergent or switch to a different blocking reagent.
|Poor binding of proteins to membrane.
|Use a membrane with the correct binding capacity. Dry PVDF membranes after transfer to promote strong binding.
|The protein in the species tested is not recognized by your primary antibody.
|Run a positive control. Check literature or perform a BLAST alignment to see whether your antibody should react with the target protein.
|Insufficient washing or blocking.
|Increase blocking time or consider using an alternate blocking reagent. Increase the number of washes.
|Concentration of the primary antibody is too high.
|Determine optimal antibody concentration through titration. Use a more dilute antibody with longer incubation times (slow targeted binding is best).
|Secondary antibody binding non-specifically, or binding with blocking agent.
|Run a secondary control with no primary antibody.
|Decrease the amount of protein loaded on the gel, or dilute the sample.
|Contamination of equipment or reagents.
|Replace reagents, ensure all equipment is properly cleaned.
|Membrane causing high background.
|PVDF membranes are considered to give higher background than nitrocellulose membranes.
|Membrane dried out during incubation.
|Ensure membrane is not drying out during the incubation period.
|Incubation temp too high.
|Incubate membrane at 4°C.
|Cross-reactivity of phospho-specific antibodies with blocking agent.
|The user may have to try different blocking buffers, such as milk, BSA, etc. to reduce non-specific binding while maintaining specific signals. For optimal results, follow the blocking buffer recommendations from your antibody provider.
|Multiple or non-specific bands
|Primary antibody concentration too high.
|Decrease the concentration of primary antibody. Run secondary control without the primary antibody.
|Excess protein on gel.
|Reduce the amount of protein loaded.
|Issues with blocking.
|Optimize blocking time and blocking reagent.
|Increase number of wash steps.
|Antibody not properly purified.
|Use antibodies purified by the affinity method.
|Target protein has been degraded.
|Use fresh sample. Include protease inhibitors in your sample buffer.
|Frequently passaged cell lines accumulate differences in protein expression profiles.
|Retrieve and expand original cell line, run samples in parallel.
|Target protein has several modified forms (acetylation, methylation, glycosylation etc.).
|Refer to literature, use agent to remove modifications when possible/necessary.
|Protein subtypes have different molecular weights.
|Use bioinformatics analysis and review literature to estimate the correct protein size.
|There are splice variants from the same protein family that share similar epitopes.
|Check literature and/or perform a blast search to confirm.
|Multimer formation of target protein.
|Prior to SDS page, boil protein for 10 min to disrupt multimers.
|Concentration of antibody too high.
|Reduce antibody concentration.
|Protein transfer too rapid or gel became over-heated during electrophoresis.
|Increase the transfer time and/or run gel at 4°C
|Too much protein loaded on gel.
|Decrease the quantity of protein loaded on gel.
|Smile effect on bands
|Migration was too rapid.
|Decrease voltage when running gel.
|Temperature during migration was too high.
|Run gel at 4°C.
|Uneven staining of gel
|Bacterial contamination of antibodies.
|Store antibodies as recommended by your antibody provider. Use fresh buffers.
|Insufficient antibody volume.
|Ensure that the membrane is completely covered with antibody and incubate with agitation.
|Target band is extremely high/low on blot
|Separation during electrophoresis not efficient.
|Change gel percentage: use a lower percentage for large proteins, a higher percentage for small proteins.
|Lane with protein ladder is black
|The antibody is reacting with the protein ladder.
|Load gel so there is a blank lane between the ladder and the first sample lane.
|Uneven white spots on blot
|Air bubbles were trapped between the gel and membrane during transfer.
|Ensure air bubbles are removed when preparing for transfer.
|Black dots on the blot
|Antibodies are binding to blocking agent.
|Filter blocking agent.