[Arabidopsis] Strep-II tag in Arabidopsis

Mary Li via arab-gen%40net.bio.net (by junyan.li from anu.edu.au)
Tue Mar 2 19:48:39 EST 2010


Hello everyone,

 

I got some questions about protein tags in Arabidopsis. I've tagged my gene
of interest with Strep-II and generated relative transgenic lines. I've done
qRT-PCR to measure the transgene level to make sure it is expressed highly,
and all transgenic plants showed strong phenotypes compared to wild type. I
tried to detect my protein on Western blot with Strep-Monoclonal Antibody
conjugated with HRP (StepMAB-HRP, from IBA), and had no luck with it because
somehow the antibody cross-reacts with most of proteins which leads to
strong unspecific binding. Then I switched to Strep-Tactin-HRP (from
BioRad), the specificity seems better than the antibody but I cannot detect
my protein of interest. This protein should be quite abundant in the plants
as we fused its promoter with GUS reporter before, and the GUS staining
shows up within 20 mins throughout the plants. Some people told me that
Strep-II is notorious for its low sensitivity to be detected, and
recommended trying to column purify the protein with Strep-Tactin first as
described in Witte et al., 2004
(http://www.springerlink.com/content/l5x2557113472017/) before doing any
Western blot.   My other alternative is try to tag this protein with HA,
which acclaims to be more sensitive and specific. I am really confused at
that moment, wondering if anyone who had similar experience can give me some
suggestion? Thank you very much in adavance.

 

Mary Li

 

PhD Candidate

BLD 41 Linnaeus Road Biochemistry and Molecular Biology

Research School of Biology

Australian National University

Canberra ACT 0200

Australia

 

Phone: (02)6125 2693

Mobile:0432845961

 

 



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