SDS-PAGE gel and running buffer - (Apr/10/2012 )
We are running multiple SDS-Page gels, but are limited with how many transfers can be run. We can run 4 gels at a time but are only able to transfer 2 at a time. I've been reading to not let the gels sit in transfer buffer for too long, but do you think we can let them sit in the running buffer (approximately 1.5hrs) while two of gels transfer without causing harm to the proteins?
Thanks in advance!
soaking in buffer for any appreciable amount of time will allow the proteins to diffuse.
you can try keeping the gels on the apparatus with a low current to maintain band sharpness or find a way to transfer all four gels at the same time (depends on type of transfer apparatus and how many are available).
I agree with the previous advice about not leaving gels in transfer buffer for such a long time.
From what you say, I am assuming this is a semi dry blotter that only takes two blots at time. Why not polymerise 4 gels at the same time but rune only 2 at atime. This way you could be running the second set at reduced voltage whicl the forst set are transferring. You could still run 4 gels and 4 blots in the same day.
I would recommend checking your transfer apparatus guide, or speaking with a rep- some machines will allow transfer 'stacking' in sandwiches (filter paper, gel, membrane, filter paper, gel, membrane, filter paper), but this often depends on a) your baseline transfer efficiency, and the specs of your machine.