I am trying to IP my protein of interest and probe for Brg1, which is a part of the SWI/SNF chromatin remodeling complex. I tried to prepare whole cell extracts from HEK-293 T cells using RIPA buffer but was not able to detect any Brg1. I am concerned that I am not extracting tightly associated nuclear proteins given that the antibody has been known to detect endogenous levels of Brg1 in HEK 293 cells. Not surprised that I was not able to co-IP Brg1.
Can someone suggest a protocol please?
Use urea in your lysis buffer. Our laboratory also works on BRG1 and we do IP for BRG1 very often. Here's the buffer composition if you might want to try: Urea lysis buffer (90% 8.8 M urea, 2% 5 M NaH2PO4 and 8% 1 M Tris-Cl; pH 8.0). All the best!
Hi,
Use of Na-Desoyxycholat might help to get the proteins out of the nucleus (causes the membrane proteins to become more soluble). If you need mainly pure nuclear extracts you could think about a sucrose cushion to seperate the nuclei from the rest of the cells before extracting the nuclear protein fraction.
Regards
Use urea in your lysis buffer. Our laboratory also works on BRG1 and we do IP for BRG1 very often. Here's the buffer composition if you might want to try: Urea lysis buffer (90% 8.8 M urea, 2% 5 M NaH2PO4 and 8% 1 M Tris-Cl; pH 8.0). All the best!
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