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RE: Protein kinase C activity assay? Help me!



Awasthi

You may want to use a better PKC substrate such as the short peptide named
"Kemptide". This peptide is a very good substrate, and it is suitable for
P-81 binding kinase assay.
Hope it will help, Yair

Yair Doza
__________________________________________________________
AstraZeneca R&D Charnwood
Discovery BioScience, Bakewell Road, Loughborough, Leics LE11 5RH,
England
Tel: +44 (0)1509 645481   Fax: +44 (0)1509 645557
yair.doza@charnwood.gb.astra.com



-----Original Message-----
From: awasthi [mailto:awasthi@uthscsa.edu]
Sent: 23 August 1999 18:19
To: kinases@sdsc.edu
Subject: Protein kinase C activity assay? Help me!


Dear kinasers,

I have been doing radioactive PKC activity assay in crude extracts of
membrane 
and cytosolic fractions of cells, using MBP 4-14 as substrate. I have been 
getting high basal activity in the membrane fraction and that obscures the 
induction of PKC activity by growth factors. The maximum increase I get is 
doubling the basal activity. What may be the reason when literature is full
of 
reports showing induction many-fold? Also, I  get the induction only when 
assay is done on membrane fraction and cytosolic fractions separately, why? 
There are many more questions I will like to ask once I get response to
these. 
Details of the method may be important to answer, probably, which I will 
provide on response.

thanks

Awasthi
UTHSCSA, San antonio
210 567 4341

UTHSCSA, 7703 Floy Curl
San Antonio, TX 78284
Ph: 210 567 4341 (O)
    210 614 2637 (H)