Removal of E.coli non-specific binding
hroychow at NMSU.EDU
Fri Jan 3 11:05:52 EST 1997
At 07:59 PM 12/30/96 GMT, DAVID PULFORD wrote:
>I'm looking for a quick and easy way of removing E.coli antibodies from a
>rabbit serum. I've found pseudoscreening an incomplete, time consuming and
>expensive way of removing this background activity for screening an E.coli
>expression library. I chanced upon a method a while back that used
>formalised cells but have no reference for this approach. Can anyone help?
This following protocol has worked for me nicely:
1.grow E. coli (500mL) overnight as usual in LB.
2.resuspend the cells in 1 mL of TBST.
3.sonicate 4x3sec bursts at full power, and at 0 C.
4.freeze the sonicated cells at -20 C for 1-2h (or O/N).
5.resonicate the thawed cells as before.
6.the resultant cell lysate is then made 0.02% in Na-azide to a final vol. of 13mL (approx), mixed by gentle vortexing and divided into three 4.3mL aliquotes.
7.incubate the rabbit IgG with these three fractions successively for 3-4h (the final incubation may be done O/N, and the volume ratio is not critical), each time centrifuging the mixture (15k/15min) before adding the next aliquote of E. coli suspension. Finally centrifuge and store the pre-absorbed anti-serum/IgG at 4 C.
This takes a day-and-a-half, and produces very clean serum/IgG. There may be other commercially available premade columns etc., but I am not aware of those.
Hope this helps,
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
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