Best IP (or co-IP) protocol for B-cells - (Oct/05/2005 )
B-cells are a beast to work with. They are chock full of immunoglobulins and present a - uh, unique- challenge to IP.
I wanted to survey y'all to see if you have a good IP protocol (for depleting all sorts of Ig or whatnot) for B cells (cell lines, primary B splenocytes, hybridomas).
Some things I'm worried about:
* How to get rid of human IgM, mouse IgM from the cell lysates (these Ig do not bind well to Protein A or G)
* Best ways/detergents to separate nuclear vs. cytoplasmic. (ranges in concentrations would be good too, kits are appreciated too but I like to know what's in it, we're an economical lab)
* Anybody IP with a chicken antibody?
* How much Ig do they make anyways?
* are you ever worried that you might saturate your reducing agent (when finally doing western) with all those disulfide bonds?
appreciate your detailed thoughts and protocols.
I used to work with B-cell lymphoma cell lines (Burkitts, LCLs). My boss frequently used to do IPs but would cross-link his monoclonals to Protein A-agarose beads before the IP. His method was taken from a paper in Virology by Simanis & Lane (1985 144:88-100).
1. Bind your ab to the protein A-agarose overnight at 4 degrees on a rotater (I'm guessing this was done in PBS?).
2. Incubate with freshly prepared 40mM dimethylpimelimidate.2HCl (10mg/ml?; Sigma D8388 250mg £7.40) in 0.1 Borate pH9 (Discard anything you don't use). Rotate at RT for 45mins.
3. Wash 2X in 50mM ethanolamine pH8 (Incubate 2nd wash for 30mins or more).
4. Wash 2X in 50mM Tris-Cl pH7.5.
I was using 2µg Ab per 20µl protein A-agarose (50% slurry). I think I cross-linked 40µg to 400µl of 50% slurry.
I'm sure there must be other protocols for crosslinking abs to protein A-agarose aswell. It also has the advantage that you won't get any Ig bands on your westerns.
All the best,