CNBr and phosphate groups

Bart Sefton Bart_Sefton at QM.Salk.Edu
Wed Mar 27 15:28:25 EST 1996


In article <4j6b04$nop at resunix.ri.sickkids.on.ca>, Randall Willis
<willis at gandalf.psf.sickkids.on.ca> wrote:

> Hello one and all,
> 
> Just wondering if anyone has heard anything about effects of CNBr 
> cleavage of proteins on the phosphates of phosphotyrosine, phosphoserine 
> or phosphothreonine.  We want to cleave our phosphorylated proteins and 
> the phosphate groups are critical to the success of our experiments.


There is no problem at all.  CNBr cleavage is used routinely with Src
kinases.  Joan Brugge's lab uses it, and my lab has a couple of
publications describing how we do it.  See Luo, K., Hurley, T. R., and
Sefton, B. M. (1990) Analysis of the sites in p56lck whose phosphorylation
is induced by tetradecanoyl phorbol acetate.  Oncogene 5:803-808, and Luo,
K., and Sefton, B. M. (1990) Transfer of proteins to membranes facilitates
both cyanogen bromide cleavage and two dimensional proteolytic mapping.
Oncogene 5:921-923.  Use of Tricine SDS gels help resolve small
fragments.  We now follow the procedure described in Schagger, H. and von
Jagow, G. (1987). Anal. Biochem. 166, 368-379 exactly.

Good Luck
Bart



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