The gel runs nicely, all the bands are nice and clear. I guess something happens during the transfer... the ladder looks fine but the lanes look fuzzy. After developing the blot, it's hard to see the bands, there are streaks running in the middle, or it's just not reproducible. I've tried everything I could think of: increasing digitonin, decreasing digitonin, loading more protein, loading less protein, increasing incubation time during solubilization, using less coomassie, of course I activate the membrane before transfer. I'm losing my mind. I've gotten it to work three times out of 34. And it doesn't help that my PI is getting fed up with me, and is accusing me of pretty negative things (which may be true, in which case I don't understand why he keeps me here).
Any tips?
One possible reason that the transfer is not working well is if the gel and the blot filter are not pressed against each other with sufficient force. Adding another layer or two of thick paper behind the gel and behind the blot filter may help in that case.
Adam B Shapiro I use two papers on top of the membrane and two sponges below the gel.
Did you try incubating your gel in the transfer buffer for longer time (30-60 min)? BTW, what is the composition of your transfer buffer?
Check the chemicals used for gel preparation, whether the concentration is correct, the dissolving capacity of the chemical. Next the sample preparation before loading, dilute the sample in different concentration..
Prepare your transfer buffer freshly and keep the transfer time a bit higher for larger MW proteins since you are applying a native page and probably you are analyzing the relatively large proteins. Always remove bubbles before the blotting process and prefer conditioned PVDF membrane for large proteins. It would be helpful if you can share the transfer buffer composition and an image of your problematic blots. By the way, the wet transfer is superior for large proteins if you chose the semi-dry transfer.
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