The truth is that I have had many, many problems to be able to see a specific band of this protein. My problems have ranged from bad transfers, to unknowns that I have not been able to answer. The times I have a good transfer (see image of ponceau s #1), I still fail to see defined bands once developed (see failed attemps). I have tried everything, increasing the concentration of the primary antibody, secondary, using more West Dura, cooling the transfer buffers to avoid burning (see ponceau s #2) and still nothing. Does anyone have any idea what else I could try? Thank you all very much in advance.
Hi Diego,
Do you put SDS in your transfer buffer? If not, then put a bit SDS (0.1% at least) to your transfer buffer. It worked for us with 8% SDS PAGE.
Thanks,
Debanjan
Hi Dr. Debanjan Mukhopadhyay
I actually don't have SDS on my transfer buffer, but both of my gels (both the concentrator and separator) do have SDS on them. Could you explain why having SDS on the transfer buffer would help?
Thanks you in advance,
Adding SDS (final concentration of 0.1%) to the transfer buffer will decrease the chances of in-gel protein precipitation.
Methanol tends to remove SDS from proteins, so reducing the percentage of methanol to 10% or less will also help against protein precipitation.
Run your samples in a low-concentration gel, 8% or less.
Use wet transfer (4 ℃ overnight) instead of semi-dry transfer.
See, for example (ignore the commercial parts):
https://advansta.com/six-tips-for-transferring-large-proteins-for-western-blotting/
https://www.bioradiations.com/six-tips-for-efficient-transfer-of-your-proteins/
Hi Diego,
large proteins tend to precipitate in gels and can't do so in the presence of SDS as Dr. Strehl mentioned. Also, I prefer wet transfer for everything. You can use 20V for overnight or 70 volts for 2-3 hrs using BioRad transfer tanks.
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