Advice Regarding Antibody Preservation

[Ryanc]

I am currently working with human cell lines (epithelial and t-lymphocyte) as a model system to create a method to extract DNA and isolate antibodies.

I am currently using a simple RIPA detergent buffer which works very well, however when I tr to preform the extraction with 20 million cells or more a snotty, colloidal mass forms and my results begin to decline.

I have determined the glob to be mostly a mass of hydrophobic cell bits (collagen, proteoglycans, etc) it is nearly impossible to pipette and sonication does not break it up. I found that if I retool the RIPA buffer with about 15-20%v/v of a nonpolar solvent like acetonitrile the glob mostly goes away.

The problem I am having is that the use of the solvents I have tried so far either butcher the DNA, destroy the antibodies I'm looking for, or it interferes with downstream tests. The other problem is that for enough material to preform all the required tests I will need to be able to lyse/solublize about 75-100 million cells in 1-2mL of buffer.

The bottom line is that I need a gentle chemical or enzyme tat will dissolve these globs of cellular debris while leaving the DNA and most antibodies intact.

Any advice would be greatly appreciated! Thank you!

[danfive]

RIPA is pretty weak in my opinion, but you can increase the SDS (normally 0.1%).

I would go straight to using CHAPS LYSIS buffer. 0.1% CHAPS added fresh to Tris-NaCl-EDTA.