Hello,
I am planning to IP and Ago2 protein from Jurkat cells, but unfortunately I haven't been doing an IP before. I am planning to use dynabeads protein G for my purposes and I have browsed through many online protocols though I may need some "starting conditions". How many total lysate is recommended, how many Abs etc. Can I use JUST BEADS as a negative control (without adding whole IgG, for example)?
Maybe there's some standard procedures that one must start from and ajust depending on the results?
Well, thanks to anyone who can help!
P.S. And, merry christmas just in case! ;)
is ur protein transfected??? i mean is it endogenous or exogenous??? if its endogenous you will require at least 500ug of protein lysate and about 200ug for a expressed protein.....lysis can be done by any lysis buffer (CHAPS will give better results).....run the ip for overnight....
Thank you Wasim.
It's endogenous, and I have already tried with Cell Signaling's RIPA buffer for lysis...
I've tried to start from indirect method, when you bing Ab with Ag first and THEN add beads. Doing it in RT, but really not sure if it will work this way.. (however some protocols tells me RT is fine)
What are the preferable temperatures for all the procedures? Should everything be carried out in +4?
oh yes....everything in a cold room...end over end incubation.....and RT is good but try the conventional way....
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