western blot troubleshooting starts with sequential analysis

Fig. 1. The sequential stages of the western blot process.
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The 8 basic steps of the science of Western blotting have remained the same for the last 40 years.

1. First, sample preparation: lysing and denaturing the proteins.
2. Next, gel electrophoresis.
3. Then membrane transfer.
4. After that blocking.
5. Then incubating the primary antibody.
6. And, then incubating the secondary antibody.
7. Next detection.
8. And, finally analysis.

But, while the steps haven’t changed, new methods of automation for the blocking, washing and incubating the primary and secondary antibodies have increased reliability and reproducibility in Western blot research.

Western blot troubleshooting

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However even with automation, not every blot turns out the way we’d like. As a matter of fact, Rice University has an online “Hall of Shame” that presents Western blot goofs. The site discusses the joys and “oh boys” of some of the most colorful Western blot failures. Sometimes blot failures can be attributed to bad antibodies; however, this article relates that variations in western blot assay results occur because of errors in manual processing.

So, to assist you, whether you do manual or automated processing, we’re providing this:

Western Blot Troubleshooting Guide (click to download the PDF)


Diffuse Bands

Possible Cause

1.  Antibody concentration too high

2. Excess protein on gel

3.  Protein transfer was too fast and/or the gel was over-heated during electrophoresis


  • 1. Decrease antibody concentration.

  • 2. Reduce the amount of total protein loaded on the gel.

  • 3. Increase transfer time
    Apply cooling system for electrophoresis.


Nonspecific bands

Possible Cause

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

3.  Degraded protein sample

4. Presence of new proteins or different splice variants that share similar epitopes

5. Presence of impurities with antibody

6. Formation of multi-mer on protein target

7. Formation of different protein subtypes which have different molecular weights

8. Presence of multi-modifier locus in protein

9. Primary antibody concentration too high

10. Non-specific bands caused by secondary antibody

11. Excess protein on gel

12. Insufficient washing

13. Blocking problem


  • 1. Wash blots after transfer
    Don’t use SDS

  • 2. Go back to original non-passaged cell line
    and then run current
    and original cell line samples in parallel.


  • 3. Use fresh sample
    use protease inhibitor.

  • 4. Check the literature for other reports.
    Perform a BLAST search.
    Use a cell line or tissue reported on the instructions.