I have to run a 2D sample after pull down assay using biotin labelled DNA and streptavidin beads (to isolate regulatory proteins of a DNA fragment), which buffer I should I use in pull down that will not produce streaking in 2D?
Hi Nisha,
streaking in 2D is because of salts, phenols, lipids, etc . But the main streaking problem I faced is because of salts which may be from extraction or any other buffer (composition) you use during sample processing. so first important thing to avoid streaking is Desalting. Desalting may be done by dialysis, 3KDa Amicon centrifugal tubes or desalting columns.
In amicon which buffer i should use to replace earlier one if u have some composition please suggest me
it is best to do buffer exchage by using milli Q water 3 times and then redissolve protein pellet in Rehydration buffer.
Yes. but u can also dilute rehydration buffer (2:1, milliQ : rehydration buffer) and use for buffer exchange.
Can you please tell me how much elution buffer you used to elute the proteins from the beads ?
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