Immunoprecipitation troubleshooting - (Jan/09/2008 )
Hi,
I am working on a protein that is not readily solubilized in RIPA.So I add urea to RIPA in order to solubilize the protein.Can anyone tell me whether I can use this urea lysate for IP.And how much should I dilute the urea so that it is compatible with immunoprecipitation by protein A/G beads?
Thanks in advance,
pparag
-ppatward-
QUOTE (ppatward @ Jan 9 2008, 09:42 AM)
Hi,
I am working on a protein that is not readily solubilized in RIPA.So I add urea to RIPA in order to solubilize the protein.Can anyone tell me whether I can use this urea lysate for IP.And how much should I dilute the urea so that it is compatible with immunoprecipitation by protein A/G beads?
Thanks in advance,
pparag
I am working on a protein that is not readily solubilized in RIPA.So I add urea to RIPA in order to solubilize the protein.Can anyone tell me whether I can use this urea lysate for IP.And how much should I dilute the urea so that it is compatible with immunoprecipitation by protein A/G beads?
Thanks in advance,
pparag
You may want to dialyze out the urea with several changes of the buffer without containing any urea. The protein should refold better without any urea, although it's not for sure that the protein would refold properly. For more details on immunoprecipitation, here is an immunoprecipitation protocol: Immunoprecipitation Protocol
-keithbrent2000-