Bacterial labelling with antibodies

[Carol Bayles]

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This is not a confocal question per se, but I am hoping someone can help.  I have a user who wants to study the localization of a His-tagged protein in E coli.   He would like to do it without tagging the protein with GFP, only using anti-His tag antibody FITC.  Can this be done?  Are there special treatments for the  cell wall  to allow the antibody to reach the protein inside the E coli?  I googled a lot, but could not find a reference to this specific method.

Thanks a lot.
Carol Bayles
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Carol Bayles, Manager
Microscopy and Imaging (MIF)
Life Sciences Core Lab Center
B46 Weill Hall
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Nanobiotechnology Center (NBTC)
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[Lloyd Donaldson]

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Carol

Do the bacteria have to be alive. Otherwise TEM and use anti-His with gold should work well avoiding the problem of getting something through the cell wall.


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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Microscopy and Imaging Facility
Sent: Wednesday, 28 November 2012 5:41 a.m.
To: [hidden email]
Subject: Bacterial labelling with antibodies

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To join, leave or search the confocal microscopy listserv, go to:
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*****

This is not a confocal question per se, but I am hoping someone can help.  I have a user who wants to study the localization of a His-tagged protein in E coli.   He would like to do it without tagging the protein with GFP, only using anti-His tag antibody FITC.  Can this be done?  Are there special treatments for the  cell wall  to allow the antibody to reach the protein inside the E coli?  I googled a lot, but could not find a reference to this specific method.

Thanks a lot.
Carol Bayles
:=:=:=:=:=:=:=:=:=:=:=:=:=:=:=:=:=:=:=:=:=:
Carol Bayles, Manager
Microscopy and Imaging (MIF)
Life Sciences Core Lab Center
B46 Weill Hall
607-254-4860
[hidden email]
http://cores.lifesciences.cornell.edu
Nanobiotechnology Center (NBTC)
www.nbtc.cornell.edu
Cornell University
Ithaca NY 14853



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[Elke Kuster-Schock]

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Hello Carol,

do I understand correctly that the researcher wants to use an antibody to see
the localization of a protein in *living* bacteria, or are they looking for an
immunostaining protocol?
In the former case, I'm not sure it can be done. My guess would be that the
bacteria would have the antibody for lunch, even if you got it across the cell
wall without seriously altering their physiological state.
For immunostaining fixed bacteria, there are a bunch of labs that have done
groundbreaking work starting in the '90s, along with developing fluorescent
protein methods for live imaging. As a starter, look up Kit Pogliano's
publications; any cool bacterial image in the Molecular Probes catalogue is
likely from her lab.

Regards,
Elke

--------------
Dr. Elke Küster-Schöck
CIAN (Cell Imaging and Analysis Network)
Proteomics & Genomics Coordinator, Microscopy Associate
McGill University
Montreal, QC, Canada
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