Re: [Fwd: kinases Acid-denaturing enolase]

[ouri <ouri@synergix.co.il>]

you wrote:

>                                                   ------------------------------------------------------------------------
>
> Subject: kinases Acid-denaturing enolase
> Date: Thu, 09 Dec 1999 11:33:16 -0500
> From: Alan Howe <alanhowe@med.unc.edu>
> To: kinases@SDSC.EDU
>
> Could anyone out there provide the/a protocol for acid-denaturing enolase
> for Src kinase assays?
>
> Thanks, in advance, for your help.
>
> P.S. Thanks to everyone that responded to my earlier PKA IP kinase assay
> question. I think I've got it working!
> ---------------
> Alan K. Howe, Ph.D.
> University of North Carolina at Chapel Hill
> Lineberger Comprehensive Cancer Center
> Dept. of Pharmacology, CB# 7365
> Mary Ellen Jones Bldg, Rm 920
> Chapel Hill, NC   27599-7365
> (919) 966-4343
> (919) 966-5640 (FAX)
> alanhowe@med.unc.edu

 Dear alan

Please find the following part of a src kinase assay protocol concerning enolase denaturation:

Enolase (from rabbit muscle - SIGMA #E0379) comes in crystalline suspension in 2.8 M (NH4) 2SO4 .  The enolase has to be acid
denaturated!!!  Resuspend to 2.5 mg/ml and dialyze against water overnight.  Add equal volume of 50 mM acetic acid (100% is
equal to 17.6 M) and incubate for 5 min. at 30oC.  Then, add 1/4 volume of 1 M HEPES (pH=7).  The concentration will be 1
mg/ml. Store in frozen aliquots.

Good luck!!

--
Ouri Fischel, Ph.D.
Synergix Ltd.
Technology Park Malha, Building 1
Jerusalem 91487
ISRAEL

tel: 972 2 6480397
fax: 972 2 6480399
email: ouri@synergix.co.il