step-wise dialysis? - (Oct/15/2009 )
bluebird on Oct 22 2009, 04:49 PM said:
even if the protein soluble in 8M urea? do you think the SDS will give a good result???
yes. the only possible problem with urea is that when you heat the sample in sds and reducing agent, the urea will decompose and may carbamylate the protein, altering the mobility.
-mdfenko-
bluebird on Oct 22 2009, 06:11 PM said:
Hi Swanny,
you didn't answer my question, how can Iadd the denatured protein drop-wise into the buffer, practically I don't have any Idea about this type of diialysis?
2_also can I run SDS at this stage for solublized protein or I have to refold it first?
you didn't answer my question, how can Iadd the denatured protein drop-wise into the buffer, practically I don't have any Idea about this type of diialysis?
2_also can I run SDS at this stage for solublized protein or I have to refold it first?
Sorry, brain must have gone to sleep. Just use a pipette to dribble the protein in. It's not actually dialysis, just an efficient way to get rid of the urea without risking unfavourable interactions leading to aggregations. You end up with a litre or so of very dilute, but hopefully correctly folded protein. You then need to concentrate and purify normally. If you have an ion exchange protocol, for example, you can refold the protein in your starting buffer and just do a bulk loading onto the column, switch over to buffer without protein to do the washing, and elute in salt.
Of course, since you have come from an inclusion body, your protein should already make up 80 - 90% of the total protein content.
-swanny-