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Re: In Gel KAs

Dear Scott,

no idea about your in-gel kinase assays but a comment about your
"kinaseology" nomenclature.

Some protein kinases are autoinhibited by a mechanism in which part of
their structure, termed
a pseudosubstrate, occupies the active site. For your edification, read
Kemp, B. E., M. W. Parker, et al. (1994). "Substrate and pseudosubstrate
interactions with protein kinases:  determinants of specificity." Trends in
Biochem. Sci. 19: 440-443.

I think what you mean't to say was "it will use myelin basic protein,
casein, or histone H1 as a
non-specific substrate" ;-)


>I've got a partially purified kinase activity.  I know this kinase will
>either ATP or GTP as its gamma p donor (is gtp usage uncommon?).  I also know
>that it will use myelin basic protein, casein, or histone H1 as a
>pseudo-substrate.  I have been attempting to get a molecular weight of this
>kinase through in gel kinase assays, but have been unsuccessful.  Does anyone
>have a good protocol, or any tips for optimizing in gels?  How about a good
>positive control?  I've tried casein kinase II alpha for that without
>luck...any info is much appreciated.
>Scott Gridley


Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy  3065  Victoria

Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676

Email: k.mitchelhill@medicine.unimelb.edu.au

Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org