CDK6 kinase assay

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Wed May 14 23:44:02 EST 2008


Hi,
check this out

*In Vitro Kinase Assay
(**33*<http://www.pnas.org/cgi/content/full/96/23/13197?ck=nck#B33>
*).* NRK (5 × 106) and 5 × 105 K6D1 cells were lysed with 2 ml and 0.5 ml of
ice-cold immunoprecipitation buffer consisting of 50 mM Hepes (pH 7.5),
150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 21 mM DTT, 0.1% Tween-20, 10% glycerol,
1 mM PMSF, 4 mg/ml each of leupeptin, pepstatin, and aprotinin, 0.1 M NaF,
2 mM sodium orthovanadate, and 10 mM [image: beta]-glycerophosphate,
respectively. Lysates were incubated at 4°C for 2 hr with 0.3 µg of [image:
alpha]Cdk4, [image: alpha]Cdk6, or [image: alpha]Cdk2. Protein G-Sepharose
suspension (15 µl) (Amersham-Pharmacia) then was added and incubated at 4°C
for an additional 1 hr. Immune complexes bound to Protein G-Sepharose were
precipitated by centrifugation and washed with ice-cold, glycerol-free
immunoprecipitation buffer. The immunopurified Cdk4, Cdk6, and Cdk2 were
incubated at 30°C for 30 min in 10 µl of reaction buffer (50 mM Hepes, pH
7.5/1 mM EGTA/10 mM KCl/10 mM MgCl2/1 mM DTT) containing 5 µg of truncated Rb
(QED Bioscience, San Diego) and 10 mM ATP. The reaction products were
electrophoresed on 10% SDS-polyacrylamide gels and transferred to
poly(vinylidene difluoride) membrane filters. The Rb molecules phosphorylated
by Cdk4 or Cdk6 were immunodetected with the anti-Ser-780-phosphorylated Rb
antibody (MBL), and the Rb phosphorylated by Cdk2 was immunodetected with
the anti-Ser811-phosphorylated Rb antibody (New England Biolabs).
http://www.pnas.org/cgi/content/full/96/23/13197?ck=nck#F5

Sudheendra.

On Wed, May 14, 2008 at 11:30 PM, Debashish Das <debashish.das from ki.se> wrote:

> Dear all
>
> I would need your help and all suggestions for doing a kinase assay
> in-vitro to detect the
> activity of CDK6. I have never done a kinase assay and as I see the papers
> published, they
> have all used only radioactive detection method!!. It would be of great
> help if anyone around
> has had experience in kinase assay preferably in looking for the CDK6 or
> similar kinase
> activities.
>
> all help and suggestions are highly appreciated and I would be deeply
> grateful for them
>
> a protocol and also if possible a source from where to get the reagents can
> help a lot more..
>
> thanking you all for your time and help
>
> best of things
>
> deb
>
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