I'm relatively new to Western Blot and I recently blotted for pCREB vs CREB in my samples. When I showed the blot to my PI, she asked what these background bands were in my blot, as she'd never seen them in blots previously. We use chemiluminescence for our Blots, and the dark bands are my expected signal. I had Ponceau stained and stripped the blot already, is it possible that some residual Ponceau is causing an issue? I'm not really sure what else it could be.
Update: Hello all, I tried extending the block time and doing extra rinses after each step but especially the ponceau staining. It seems to have solved the issue so thank you everyone! Note: I did stain with a different antibody as this was for a different experiment so I wasn't sure if changing the primary antibody concentration would help.
- It might be due to increased concentration of the antibodies. Try to decrease the concentration of primary antibody. When the concentration is very high, then the substrate is consumed very quickly, hence very little light is absorbed leading to a white band after detection.
- Perform careful washing after primary & secondary antibody in TBST.
- Use freshly prepared ECL detection solution.
They seem to me like precipitated ponceau dye.
You can try the following:
1. shake the ponceau dye very well to ensure there are no precipitates
2. wash a bit longer than you previously did
I hope this helps. Good luck
For me it seems that you can see the shadow of the rest of proteins in your electrophoresis. In other words, in these regions of your blot the membrane is completely blocked. This means that you have background in the rest of the membrane resulting in these clear shadows. Try to block better with a higher amount of BSA or the blocking agent that you are using, or extend the blocking time.
Thank you for the suggestions, I'll try these with my next blot and share how it looks.
Hi Ashley,
You can check for the antibody dilutions too and increase the dilutions appropriately.
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