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

Proceed as with western blot.

Works fine for me ;-)


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

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