Download amino acids M 1 - Cell Signaling Technology

This enzyme was purified and extensively validated in a radioactive filter binding assay by
a collaborator of Cell Signaling Technology. Cell Signaling Technology is in the process of
revalidating this reagent in house using several technologies relevant to the screening
community including antibody based detection methods.
Datasheet: CDK2/CycE
Purified Protein (Western blot)
Description: Human CDK2 Full Length
c-DNA (GenBank accession No. X62071),
amino acids M1 – L298.
Human Cyclin E Full Length c-DNA
(GenBank accession No.M73812), amino
acids M1 – A394; both N-terminally fused to
GST-HIS6-Thrombin cleavage site
anti-CDK2
1
kDa
Theoretical MWCDK2: 60,222 Da
Theoretical MWCyclinE: 72,041 Da
Expression: Baculovirus infected Sf9
cells (co-expression of both constructs)
Purification: One-step affinity purification
using GSH-agarose
Storage buffer: 50 mM Tris-HCl, pH 8.0;
100 mM NaCl, 5 mM DTT, 15 mM reduced
glutathion, 20% glycerol
Storage temperature: -80°C
Avoid repeated freeze-thaw cycles!
Protein concentration: 0.243 µg/µl
(Bradford method using BSA [Sigma: A7638, Lot# 79H7641] as standard protein)
anti-CycE
kDa
250
250
150
150
100
100
75
75
GSTCDK2
100
GSTCycE
50
37
37
37
25
20
25
20
25
20
15
10
15
10
15
10
1: SP009, 500 ng
1: SP009, 500 ng
1
kDa
30
n=3
25
20
15
10
VMax = 29.1 pmol/µg×min
5
0
-
250
150
150
-
100
100
-
75
75
50
37
25
20
25
20
15
15
10
1: SP009, 1.0 µg
GST-CycE
GST-CDK2
50
37
1: SP009, 500 ng
Kinase activity:
250
GST-CycE
GST-CDK2
GST-CycE
GST-CDK2
ATP-KM = 1.36
0
1
2
3
4
5
6
7
µM
8
9
Conc. ATP (µM )
Coomassie stain
1
kDa
75
50
50
1
kDa
150
Purified protein (SDS-PAGE):
Silver stain
anti-GST
1
250
V ( pmol ATP/µg×min)
#7524
Please contact us at [email protected] for details.
-
Final assay concentrations:
60 mM HEPES-NaOH, pH 7.5
3 mM MgCl2
3 mM MnCl2
3 µM Na-orthovanadate
1.2 mM DTT
ATP (variable)
2.5 µg / 50 µl PEG20.000
Substrate: Histone H1, 2 µg / 50 µl
Recombinant CDK2/CycE: 100 ng / 50 µl
Assay: MAFC (Millipore) filter assay
1: SP009, 2.0 µg
Specific activity: 29 pmol/µg min
Orders n 877-616-CELL (2355)
[email protected]
Support n 87-678-TECH (8324)
[email protected]
Web
n
www.cellsignal.com
10