Immunoscreen on colonies
roney.graf at uni-konstanz.de
Thu Feb 18 07:31:50 EST 1999
In article <36CB8264.A2EB68C4 at itsa.ucsf.edu>, Thierry Giffon
<giffon at itsa.ucsf.edu> wrote:
> Hi everybody,
> I'm trying to find a protocol for a Immuno screening of E.Coli colonies.
> I want to induce the expression of a protein X with IPTG on filters, and
> treat the filter for a antibody screening.
> Does anybody knows about a protocol for this ?
> I found a reference to Current Protocols in Molecular Biology, but after
> reading the chapter twice, I gave up: Everything is for lambda GT11
> plaque screening. I don't know if a regular lysis (NaOH, NaCl) as used
> for treatment for hybridization with a DNA probe will do the trick.
> Thanks if someone can give me a reference for this.
Right, I just combined the first part of a colony (DNA) hybridization
protocol with the second part of a western: alkaline lysis on-filter on
0.5M NaOH/1.5M NaCl, neutralization on 1M tris.Cl 7.5. Then wash, block,
etc like a western.
My only problem was a considerable background. As a workaround, I tried
to capture as little cell material as possible by lifting the filter off a
velvet replica (well, I didn't have to induce expression on the filter).
By including reliable positive and negative controls, I could stop the
color reaction (AP/BCIP/NBT) at the most useful point. Different
strain/protein/antibody combinations might look cleaner.
An alternative lysis technique using chloroform vapor is described in
Maniatis (section 12.23ff).
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