Question:
I’m experiencing a persistent issue with my Western blot experiments: I consistently get completely black membranes (no visible bands at all, not even non-specific ones) after developing the blot. I’ve repeated the experiment several times and tried various optimizations, including:
- Changing the primary and secondary antibodies
- Adjusting the transfer conditions (time, voltage, buffer composition)
- Confirming protein loading and running controls
Despite all these efforts, I still get no signal. What could be the possible causes of this issue? I would greatly appreciate any suggestions for troubleshooting.
Hi, how are you? 😊 I hope you could share an image or photo of what you're seeing. I don’t have much experience with Western blotting myself, but I suggest adjusting the gel percentage (between 10–15%) depending on the size of your protein.
Also, are you using the same protein samples repeatedly (previously extracted ones), or do you extract fresh samples each time? It might be that your protein is degrading during the extraction process. Another possibility is the nature of the protein itself, especially if no one has worked on it before
similar to what is mentioned above, in addition to sharing a photo of the blot, if you could edit your first post and include the step-by-step protocol of how you performed the gel electrophoresis and WB, that could help others troubleshoot this problem with you.
Hi Sana,
What kind of membrane are you using to transfer the protein onto?
Thank you,
Hediye.
I really appreciate your input and responses.
Yes, I’ve been experimenting a bit with the gel percentage depending on the protein size (12.5% gel). And a good point about the samples—I tested both the old and the new samples, but the result was the same. Moreover, the proteins I am working on are well-known and not new.
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