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Overcome your Western blot difficulties with our troubleshooting advice, covering problems such as weak/no signal, non-specific bands, high signal, and other common issues.
Titrate the antibody to determine optimum concentration.
The antibody may have lost activity – perform a dot blot to determine activity and optimal concentration.
Include a positive control (e.g., overexpressed protein, purified protein, positive cell line, etc. Adjust protein loading accordingly).
Change incubation time and temperature (4°C, overnight).
Load more protein per well.
Enrich low-abundance proteins by immunoprecipitation, fractionation, etc.
Use appropriate treatment to induce target protein expression or modification.
Ensure sample has not degraded.
Include protease inhibitors in the lysis buffer.
Use the optimum lysis buffer for the target protein’s subcellular localization.
Check protein loading with an internal loading control antibody.
Select PVDF or NC membranes based on hydrophobicity/ hydrophilicity of the target antigen.
Blocking for too long can mask specific epitopes and prevent antibody binding.
Reduce the percentage of, or remove, the blocking reagent from the antibody incubation buffers.
Switch to using an alternative blocking reagent.
Use a Tris-tricine gel for protein targets <20kDa.
Reduce transfer times and/or use smaller pore size membranes (0.22 μm) for low MW proteins <30kDa.
Wet transfer is recommended for small proteins (10kDa).
Ensure proper transfer set-up (e.g., no air bubbles trapped between the gel and the membrane).
Thicker gels can result in incomplete transfer of high molecular-weight-proteins.
Check the quality of protein transfer with a reversible, universal protein stain, e.g., Ponceau-S.
Wet transfer produces higher-resolution transfers over semidry transfer.
The presence of sodium azide inhibits the activity of HRP.
Use sodium azide-free buffers.
Ensure sufficient washing.
Check several exposure times to achieve optimum detection.
Try different detection reagent compositions and/or brands.
Dilute chemiluminescent reagents in high-purity water.
Too much primary and/or too much secondary antibody.
Use antibodies at higher dilutions.
The antibody reacts with the MW marker.
Migration through the gel was too hot or too fast.
Reduce the voltage applied to run the SDS-PAGE gel or run the gel in a cold room.
High protein concentrations can result in diffuse protein bands.
Uneven protein loading: assay protein samples and load by protein amount. Check for even protein loading by stripping and reprobing the blot with an internal control antibody (or use an HRP-conjugated loading control antibody).
Uneven gel composition (gel has set too quickly while casting or buffer was mixed inadequately).
Uneven bands can be due to insufficient buffer being added to the tank during running.
This problem can be caused by antibodies binding to the blocking reagent in the blocking buffer.
Change to another blocking reagent.
Filter the blocking buffer.
Wash excess detection reagent from the membrane before exposure.