Western blots

Sebastian Bunka bunka at i112pc09.vu-wien.ac.at
Wed May 7 03:23:34 EST 1997


In article <e-anderson-0605970936540001 at news.ski.mskcc.org>,
	e-anderson at ski.mskcc.org.nospam (eric anderson) writes:
> In article <336E29B5.211C at peakaccess.net>, Michael Tarasev
> <tarasev at peakaccess.net> wrote:
> 
>> I would like a method or information about a method of western blotting
>> involving immobilization of bacterial colonies to the blotting membrane
>> and probing for the antibody reaction directly. I have done Western
>> Blots the typical way ie running a SDS-PAGE gel and transferring the
>> proteins the PVDF membrane and carrying out the antibody reaction but
>> would like a method for using this on a smaller scale, ie in a bacterial
>> colony.
> 
> this protocol is in Current Protocols in Molecular Biology.  i've never
> used it but i stumbled across it the other day.  it's basically a colony
> screen (from a library) except that you use an Ab instead of a DNA probe
> for the hybridization.  i'm not sure if the protocol includes the use of
> PVDF membranes but that can be easily modified.
> 
It's just as easy as doing a colony blot for hybridization:
plate your bugs on a LB plate w/ antibiotic and IPTG,
incubate overnight, wit 2 forceps place a NC disk onto the LB plates
with the colonies on it. Leave on agar until the NC is completely
wet. Mark NC and agar plate by punching asymetrically thru the NC (
with a needle soaked in ink). Lift NC and move to a fresh
LB plate w/ IPTG. Colonies UP! and inkubate for 2h.
Lyse colonies in chloroform vapor for 15 minutes (place a couple
of pasteur pipettes in a glass dish and add chloroform less than 
thickness of the pipettes; place NC onto pipettes).
place in appropriate dish and add blocking solution. After blocking
SCRUB CAREFULLY OFF ANY COLONY MATERIAL WITH GLOVES ON YOUR HAND
TO AVOID FALSE POSITIVE SPOTS !!.

Proceed as with western blot.

Works fine for me ;-)

Sebastian
-- 

;-)
------------------------------------------------------------------------
Sebastian Bunka                 ph. (+43-1) 250 77 4208
Institute of                    FAX (+43-1) 250 77 4290
Medical Chemistry               email: Sebastian.Bunka at vu-wien.ac.at
University of Veterinary Medicine - Vienna - Austria
------------------------------------------------------------------------



More information about the Methods mailing list