Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
:

:

:

:

Product Information

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human CDK1 (Met1-Met295) (GenBank Accession No. NM_001786) and full-length human CycB (Met1-Val433) (GenBank Accession No. NM_031966), both with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Product Description

The kit provides a means of performing kinase activity assays with recombinant human CDK1/CycB kinase. It includes active CDK1/CycB kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine/threonine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Formula Peptide substrate, Biotin-Rb (Ser780): 2,150 Daltons. GST-CDK1 kinase: 64 kDa, GST-CycB: 78 kDa.

Background

Cyclins and cyclin-dependent kinases (CDK) are key regulators in mammalian cell cycle. Regulation of these complexes occurs through cyclin production and its destruction, relocation, inhibitory/activating phoshorylation, relocation and modification by other proteins. Each cyclin associates with one or two CDKs and most CDKs associate with one or two cyclins (1-3). CDK1 forms a complex with cyclin A/B and regulates phosphorylation of cytoskeleton proteins involved in mitosis. CDK2 and CDK3 form complexes with cyclin E, which regulate the G1-S phase transition while the CDK2/CycA complex regulates S phase progression (4,5). CDK4/CycD and CDK6/CycD are activated by mitogenic signaling during early G1 and progressively accumulate as cells transition through this phase of the cell cycle. CDK5 is activated in postmitotic neurons and regulates neuron migration during brain development (6). CDK7/CycH is believed to form a link between transcription and cell cycle. CDK8/CycC and CDK9/CycT are involved in transcription (1,2). The kinase activity of CDKs is tightly regulated by phosphorylation and protein-protein interactions. Activation of CDKs requires binding to a specific cyclin and phosphorylation of a conserved threonine residue in a region called the T loop. Examining the phosphorylation of peptides by CDK/cyclin complexes suggests that both CDKs and cyclins play a role in recognizing substrates. A consensus sequence, (S/T)PX(R/K), is identified in the peptides that are phosphorylated by CDK/cyclins.

  1. Schang, L.M. (2002) J. Antimicrob. Chemother. 50, 779-792.
  2. Murray, A.W. (2004) Cell 116, 221-234.
  3. Chow, J.P. et al. (2003) J. Biol. Chem. 278, 40815-40828.
  4. Hofmann, F. and Livingston, D.M. (1996) Genes Dev. 10, 851-861.
  5. Golsteyn, R.M. (2005) Cancer Lett. 217, 129-138.
  6. Xie, Y. and Tsai, L.H. (2004) Cell Cycle 3, 108-110.
  7. Holmes, J.K. and Solomon, M.J. (1996) J. Biol. Chem. 271, 25240-25246.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、 CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、 または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、 いかなる効力も効果も有しません。

研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

Revision 1
#7519

HTScan® CDK1/CycB Kinase Assay Kit

HTScan® CDK1/CycB Kinase Assay Kit: Image 1 Expand Image
Figure 1. CDK1/CycB kinase activity was measured in a radioisotopic filter binding assay using the following reaction conditions: 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: Rb CTF, 5 µg /50 µl, recombinant CDK1/CycB: 100 ng/50 µl.
HTScan® CDK1/CycB Kinase Assay Kit: Image 2 Expand Image
Figure 3. Dose dependence curve of CDK1/CycB kinase activity: DELFIA® data generated using Phospho-Rb (Ser780) Antibody #9307 to detect phosphorylation of substrate peptide (#1142) by CDK1/CycB kinase. In a 50 µl reaction, increasing amounts of CDK1/CycB and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® CDK1/CycB Kinase Assay Kit: Image 3 Expand Image
Figure 5. Staurosporine inhibition of CDK1/CycB kinase activity: DELFIA® data generated using Phospho-Rb (Ser780) Antibody #9307 to detect phosphorylation of substrate peptide (#1142) by CDK1/CycB kinase. In a 50 µl reaction, 50 ng CDK1/CycB, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® CDK1/CycB Kinase Assay Kit: Image 4 Expand Image
Figure 4. Peptide concentration dependence of CDK1/CycB kinase activity: DELFIA® data generated using Phospho-Rb (Ser780) Antibody #9307 to detect phosphorylation of substrate peptide (#1142) by CDK1/CycB kinase. In a 50 µl reaction, 50 ng of CDK1/CycB and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
HTScan® CDK1/CycB Kinase Assay Kit: Image 5 Expand Image
Figure 2. Time course of CDK1/CycB kinase activity: DELFIA® data generated using Phospho-Rb (Ser780) Antibody #9307 to detect phosphorylation of substrate peptide (#1142) by CDK1/CycB kinase. In a 50 µl reaction, 50 ng CDK1/CycB and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)