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FLAG IP incubation time - Duration for FLAG IP (Jun/13/2008 )

Is there any difference with 1 hour 4C FLAG IP and overnight 4C FLAG IP?

Will more FLAG fusion protein bind for overnight IP?

-leyhian83-

QUOTE (leyhian83 @ Jun 13 2008, 06:15 PM)
Is there any difference with 1 hour 4C FLAG IP and overnight 4C FLAG IP?

Will more FLAG fusion protein bind for overnight IP?


It depends upon the particular flag-ab clone you have. Some Abs work as well in an hour as overnight, but generally, and largely, overnight incubation gives more binding for all antibodies.

Some Flag-ip protocols, see what they are doing: http://search.vadlo.com/b/q?sn=158621799&a...ation&rel=0

..

-cellcounter-

For an IP, one hour is a very short incubation time, especially at 4degree. The shortest incubation I've ever seen for an IP is three hours and this was for a highly purified, high affinity antibody. Most likely, more FLAG fusion protein will bind overnight but so will more background!!! For a first experiment, just try the overnight and see what you get. You can always try different washing conditions (higher and/or lower salt and more detergent) and incubation times to get rid of background but IPs can be very tricky and take much optimization. Also, if you are really having trouble with background you can pre-clear your lysates by incubating with a control IgG of the same species your primary is made in before setting up the IP. This will get rid of much of the non-specific binding.

-rkay447-