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Re: ERK kinase assay



At 11:31 02/08/1999 +0100, marianne.fjording@leo-pharma.com wrote:
>
>
>
>I have a little problem with the substrate MBP.
>
>I i.p with ERK1 (Santa Cruz) and then perform the kinase assay using MBP 
>(Sigma)
>as substrate together with P32-ATP. Then I run a 15% SDS-PAGE and perform
>quantification by phospho-imager analysis. But here is my problem - I see a
>double band of MBP - one at 20 kDa and one at 14 kDa. It actually looks like 
>the
>MBP is degraded.
>Has anyone experience something like that - can MBP be degraded during the
>boiling of the sample before loading to the gel. Has anyone tried NOT to boil
>the sample before loading the gel - or does that make any sense?!
>
>Thank you in advance
>Marianne Scheel Fjording
>LEO Pharmaceutical Products
>Denmark
>
Hi Marianne,

i actually had the same thing (also mbp, sigma) its only 50-80?% pure (not
sure bout the xact %) also: its not (what i supposed it to be)
genbank:http://www.ncbi.nlm.nih.gov/  
DEFINITION  MYELIN BASIC PROTEIN (MBP) (MYELIN A1 PROTEIN).
ACCESSION   126803
PID         g126803
DBSOURCE    SWISS-PROT: locus MBP_RABIT, accession P25274) but i got some
reference from sigma:
Arch. Biochem. Biophys., 153, 590-598, 1972

concluding: dont worry bout the double bands, just use it
good luck!
Bart


B.M.Engels, M.Sc

Leiden/Amsterdam Center for Drug Research 
Division of Medical Pharmacology
Sylvius Laboratories,  Leiden University
Wassenaarseweg 72 / 2333 AL Leiden
P.O. Box 9503 / 2300 RA Leiden 
The Netherlands

Phone +31 71 527	6219
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e-mail: b.engels@lacdr.leidenuniv.nl