Screning E.coli transformants with an antibody?
Hiranya S. Roychowdhury
hroychow at NMSU.EDU
Fri Feb 5 12:07:09 EST 1999
At 01:13 PM 2/5/99 +0000, Fergus Doherty wrote:
>Hello, I hope you can help with this question.
>I need to screen E.coli transformants rapidly and simpel for expresion of a
>recombinant protein. The Vector used provides ampicillin resistance but
>there is no blue/white selection. It is possible that I can get
>transformants that don't contain my insert (but contain plasmid without
>insert), so I need to screen a reasonable number of transformants with an
>antibody to the recombinant protein. I understand I could pick colonies
>and transfer them to a gridded agar plate, and to a gridded nitrocellulose
>filter, on top of an agar plate with IPTG to induce expression of my
>protein. Then the problem is lysing the cells in such a way as to fix the
>proteins on to the filter prior to probing with antibody.
>Can anyone sugest a protocol or tell me more about this method of screening?
1. Grow the colonies on NC membranes directly against a plate of LB
w/antibiotics. After about 6-8h growth, trasfer membrane with colonies to a
plate containing the inducer (IPTG).
(Or, transfer colonies from a plate on to the membrane and to a plate
containing the inducer)
2. Grow for 4-5h.
3. Lyse the colonies in situ with chloroform. Alternatively, lysis can be
achieved by SDS-ALkali method (see Maniatis).
4. Air-dry the membranes.
5. Proceed with blocking and detection as you would for a SDS-PAGE transfer.
Note: Certain conditions viz. level of inducer, length of induction, method
of lysis have to be worked out impirically.
ALternattive: PCR with nested primers to detect plasmids with inserts. Then
proceed to induce/express the recombinant protein in liquid cultures for
Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
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