Troubleshooting Tip 7: Western Blot doesn't work, How can I fix this?


The above image depicts a Western blot where the protein of interest is detected by chemiluminescence. Although immunoblotting is straightforward when a protocol has been optimized for a specific protein sample, antibodies, and detection system, it is very frustrating when it does not work, especially when the lab next door uses seemingly identical conditions successfully. Here we list the main points one needs to troubleshoot when Western blotting doesn't work.

Possible Causes and Solutions

Protein Sample Preparation

1. Take extra care to keep the proteins and/or the epitopes (e.g., phosphorylated residues) of interest intact when preparing the protein sample. Work on ice, include protease and/or phosphatase inhibitors in the lysis buffer, and heat the sample immediately after lysis.

2. Estimate how much protein you have in your sample. The sensitivity of detection depends on the detection system chosen. If your sample is too diluted or the protein is present in trace amounts, use a detection system with high sensitivity (e.g., enhanced chemiluminescence detects picograms of protein).

Protein Transfer

1. Gel crosslinking and thickness affect transfer efficiency. 0.5- to 0.75-mm-thick gels will transfer more efficiently than thicker gels. Gels with a higher acrylamide percentage will also transfer less efficiently.

2. Use the correct orientation of membrane and gel relative to the anode and cathode. The membrane side should face the positive electrode.

3. Use prestained molecular weight protein standards to assess protein transfer efficiency visually at the end of the transfer. Use a reversible total protein stain (e.g., Ponceau S) to visually inspect the blot and verify protein transfer.

4. Remove air bubbles when assembling the transfer stack. Any bubbles in the filter paper stack or between the filter paper, membrane, and gel will block current flow and prevent protein transfer. This problem will be evident on a Ponceau S–stained membrane by sharply defined white areas devoid of protein.

5. Consider the use of SDS or/and methanol that could interfere with transfer efficiency and/or membrane binding. Adding SDS to the transfer buffer improves protein transfer but may interfere with protein binding to the membrane. Methanol improves the binding of proteins to the membrane; however, it can hinder transfer.

Primary and Secondary Antibodies

1. Make sure the primary antibodies recognize the denatured form of the protein of interest.

2. Use an appropriate dilution of primary and/or secondary antibodies. Test serial dilutions of each the primary or secondary antibody (keeping the other one constant) in order to determine the optimal concentration to use.


1. Use fresh reagents (e.g., luminol and peroxides for chemiluminescence) for the detection assay.

2. Vary exposure time to determine the optimal time needed to detect the bands of interest. The amount of protein in the sample, the dilution of primary and secondary antibodies and the detection assay will determine the optimal exposure time for each protein.

3. Use ubiquitous proteins (e.g., actin or tubulin) as an internal protein control to make sure that the detection assay works.

4. Use a detection assay that is sensitive enough for the amounts of protein you expect to have in your sample. Current chemiluminescent assay kits detect picograms of protein.

Details on how to perform immunoblotting and immunodetection can be found in Current Protocols Essential Laboratory Techniques, Unit 8.3, Current Protocols in Cell Biology, Unit 6.2, and Current Protocols in Immunology, Unit 8.10. Information on SDS-PAGE can be found in Current Protocols Essential Laboratory Techniques, Unit 7.3, Current Protocols in Protein Science, Unit 10.1 and Unit 10.3, Current Protocols in Cell Biology, Unit 6.1 and Unit 6.5, and Current Protocols in Microbiology, Appendix 3M.

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Contributed by: Manos Mavrakis.