Anyone have sucess with anti-nHis antibodies?

Mr. Chris Hoyle bmbckh at biovax.leeds.ac.uk
Tue Aug 19 05:03:50 EST 1997


In article <mdm8-1808971648030001 at 132.206.201.68>
mdm8 at musica.mcgill.ca (Stephen Morris) writes:

> Hi,  I've been trying to use several Quiagen antibodies without sucess.  I
> have been trying to detect a protein that is labeled with RGS6Histidine on
> the C-terminal end.  I can detect the protein on western with an antibody
> specific to the protein itself but cannot detect it with either
> antiRGS6His, antiTetraHis, or antiPentahis.  I've also tried to detect
> another protein that is 6His labelled and although I dotblot a purified
> aliquot I cannot detect it with either penthis or tetrahis.  I've been
> using the quiagen protocol which suggests blocking with casein. My
> secondary is antimouse HRP. Does anyone have a protocol that works or are
> these antibodies useless?
> Thanks    Stephen.

I use the anti-RGSH6 mouse monoclonal antibody from Qiagen without any
problems. I use it to detect a membrane protein tagged at the
C-terminus and expressed in E. coli. I use a goat anti-mouse IgG
conjugated to HRP and visualised using ECL from Amersham Life Sciences.
The antibody does not cross react with any E. coli proteins when whole
E. coli are lysed with lysozyme and SDS, protein is separated by
SDS-PAGE and blotted onto PVDF (Pall). Blots using the antibody are
very clean with little or no background; the only signal present is due
to the tagged protein.


------------------------------------------------------
Chris Hoyle

   Department of Biochemistry and Molecular Biology
University of Leeds             phone +44 0113 2333172
Leeds LS2 9JT                   FAX   +44 0113 2333167
Great Britain           e-mail BMBCKH at biovax.leeds.ac.uk
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