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AW: MAP kinase protocol



Hello Alex,

my protocol is

50mM HEPES 7,5
10 mM MgCl2
100 mM ortho-vanadate
1 mM DTT
40 µM cold ATP
1-5 µCi/sample 32P-ATP
0,5 mg/ml MBP (use Gibco`s MBP, it`s the best)
30 min 30°C on a shaker
then spot 20 µl on P81-Filter (Phosphocellulose)
wash 6-8 times (15 min each) with 0,75% Phosphoric-Acid (scintillation
counting of supernatant should be OK after 6 times washing)
wash filters 1x in acetone (2 min)
let them dry and measure in scintillation counter

shake the filters without magnetic stirrer !!!
if you have problems with bachground, reduce 32P-ATP or change to
33P-ATP.

The assay works nicely and is very reproducable.

Good luck

Matthias


> -----Ursprüngliche Nachricht-----
> Von:	Alex Lyakhovitch [SMTP:loally@uta.fi]
> Gesendet am:	Samstag, 3. Juli 1999 16:19
> An:	kinases@sdsc.edu
> Cc:	kinases@postal.sdsc.edu
> Betreff:	MAP kinase protocol
> 
> Hi,
> 
> Is there anybody who did MAP kinase assay with P-32-ATP, MBP, filter
> binding? 
> What buffer should I use for best results?
> 
> I used 50mM Hepes-HCl pH8.0, 2.0 mMDTT, 0.1 EGTA, 5mM Mg(Ch3COO)
> buffer but
> it seems to me that the binding system does not work.
> 
> Can anybode give me a good hint why it could happen?
> 
> Thanks in advance,
> Regards,
> Alex
> loally@uta.fi
> 
> Alex Lyakhovich, M.D.
> University of Tampere
> Medical School
> Box 607 
> FIN-33101 Tampere
> Finland
> --------------------
> tel. 358-3-2156640
> fax 358-31-156170