Revision 1

#9932Store at -20C

1 Kit

(8 x 20 microliters)

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 Includes Product # Quantity Mol. Wt Isotype/Source
CDK2 (78B2) Rabbit mAb 2546 20 µl 33 kDa Rabbit 
p27 Kip1 (D69C12) XP® Rabbit mAb 3686 20 µl 27 kDa Rabbit IgG
Cyclin D1 (92G2) Rabbit mAb 2978 20 µl 36 kDa Rabbit IgG
CDK6 (DCS83) Mouse mAb 3136 20 µl 36 kDa Mouse IgG1
Cyclin D3 (DCS22) Mouse mAb 2936 20 µl 31 kDa Mouse IgG1
p21 Waf1/Cip1 (12D1) Rabbit mAb 2947 20 µl 21 kDa Rabbit IgG
CDK4 (D9G3E) Rabbit mAb 12790 20 µl 30 kDa Rabbit IgG
p18 INK4C (DCS118) Mouse mAb 2896 20 µl 18 kDa Mouse IgG2a
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl Horse 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

Cell Cycle Regulation Antibody Sampler kit offers an economical way of detecting eight integral cell cycle regulation proteins. The kit contains enough primary and secondary antibodies to perform two western blot experiments with each primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

Eukaryotic cell cycle progression is dependent, in part, on the tightly regulated activity of cyclin dependent kinases (CDKs). Cyclin D/CDK4/6 activity occurs in mid-late G1 phase, upstream of CDK2/cyclin E activity. Both of these activities are required for hyperphosphorylation of the retinoblastoma gene product (pRb). pRb phosphorylation allows the release of S phase-promoting transcription factors and is indicative of the cell's commitment to proliferate. This point in the cell cycle is known as the restriction point. Cyclin protein levels oscillate throughout the cell cycle, and their availability is a means of controlling CDK activity and cell proliferation. Cyclin D is degraded through the ubiquitin proteasome pathway in the absence of mitogenic signaling. Ubiquitination of cyclin D1 is enhanced by phosphorylation at Thr286 by glycogen synthase kinase 3b (GSK-3b) (1). p27/Kip1, p57 Kip2 and p21 Waf1/Cip1 are members of the Cip/Kip family of cyclin-dependent kinase inhibitors. They form heterotrimeric complexes with cyclins and CDKs, inhibiting kinase activity and blocking progression through G1/S phase (2). However, p21 may enhance assembly and activity of cyclin D/CDK4/6 complexes (3). Levels of p21 and p27 protein are controlled through ubiquitination and proteasomal degradation (4). Levels of p27 are upregulated in quiescent cells and in cells treated with negative cell cycle regulators. p27 nuclear localization is controlled by Akt-dependent phosphorylation at Thr157 (5). The inhibitors of CDK4 (INK4) family include p15 INK4B, p16 INK4A, p18 INK4C, and p19 INK4D. All INK4 proteins selectively inhibit CDK4/6 activity, either in a binary complex, or in a ternary complex including cyclin D, resulting in inhibition of cell division (6,7).

  1. Diehl, J.A. et al. (1997) Genes Dev 11, 957-72.
  2. Pestell, R.G. et al. (1999) Endocr Rev 20, 501-34.
  3. Cheng, M. et al. (1999) EMBO J 18, 1571-83.
  4. Sheaff, R.J. et al. (2000) Mol Cell 5, 403-10.
  5. Shin, I. et al. (2002) Nat Med 8, 1145-52.
  6. Guan, K.L. et al. (1994) Genes Dev 8, 2939-52.
  7. Hirai, H. et al. (1995) Mol Cell Biol 15, 2672-81.

Background References

    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.

    Limited Uses

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    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #9932

    Cell Cycle Regulation Antibody Sampler Kit

    Cell Cycle Regulation Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1.0 mg/mL) from HeLa cells using CDK2 (78B2) Rabbit mAb #2546. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Cell Cycle Regulation Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (1.0 mg/mL) from MCF-7 cells using p21 Waf1/Cip1 (12D1) Rabbit mAb #2947. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Cell Cycle Regulation Antibody Sampler Kit: Image 3 Expand Image
    Flow cytometric analysis of Jurkat cells using p27 Kip1 (D69C12) XP® Rabbit mAb (right) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left) and Propidium Iodide (PI)/RNase Staining Solution #4087 to measure DNA content. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Cell Cycle Regulation Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using CDK4 (D9G3E) Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from HeLa, NIH/3T3, C6 and COS cells, using CDK2 (78B2) Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from HeLa, 293 and Ramos cells, using p18 INK4C (DCS118) Mouse mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from SK-N-MC, C6 and IMCD3 cells, using Cyclin D3 (DCS22) Mouse mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis from control HeLa cells (lane 1) or p21 Waf1/Cip1 knockout HeLa cells (lane 2) using p21 Waf1/Cip1 (12D1) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the p21 Waf1/Cip1 knockout HeLa cells confirms specificity of the antibody for p21 Waf1/Cip1.
    Cell Cycle Regulation Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from MCF7, L929 and C6 cells, using Cyclin D1 (92G2) Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from HeLa, IM-CD-3 and C6 cells, using CDK6 (DCS83) Mouse mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis from control HeLa cells (lane 1) or p27 Kip1 knockout HeLa cells (lane 2) using p27 Kip1 (D69C12) XP Rabbit mAb #3686 (upper) or B-Actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the p27 Kip1 knockout HeLa cells confirms specificity of the antibody for p27 Kip1.
    Cell Cycle Regulation Antibody Sampler Kit: Image 12 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Cell Cycle Regulation Antibody Sampler Kit: Image 13 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.
    Cell Cycle Regulation Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using CDK4 (D9G3E) Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 15 Expand Image
    Flow cytometric analysis of Jurkat cells using CDK2 (78B2) Rabbit mAb (right) and Propidium Iodide (PI)/RNase Staining Solution #4087, compared to a secondary only sample (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Cell Cycle Regulation Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Cyclin D3 (DCS22) Mouse mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of extracts from various cell types using p21 Waf1/Cip1 (12D1) Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Cyclin D1 (92G2) Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 19 Expand Image
    Western blot analysis of extracts from various cell types using p27 Kip1 (D69C12) XP® Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using CDK4 (D9G3E) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Cell Cycle Regulation Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Cyclin D3 (DCS22) Mouse mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 22 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® p21 Waf1/Cip1 siRNA II (+), using p21 Waf1/Cip1 (12D1) Rabbit mAb #2947 and α-Tubulin (11H10) Rabbit mAb #2125. The p21 Waf1/Cip1 (12D1) Rabbit mAb confirms silencing of p21 Waf1/Cip1 expression and α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of p21 Waf1/Cip1 siRNA.
    Cell Cycle Regulation Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded Apc (min/+) mouse intestine using Cyclin D1 (92G2) Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 24 Expand Image
    Immunoprecipitation of p27 Kip1 from 293 cells using p27 Kip1 (D69C12) XP® Rabbit mAb. Western analysis was performed using the same antibody. Lane 1 is 5% input.
    Cell Cycle Regulation Antibody Sampler Kit: Image 25 Expand Image
    Confocal immunofluorescent analysis of MCF7 cells using CDK4 (D9G3E) Rabbit mAb (green), p21 Waf1/Cip1 (12D1) Rabbit mAb (Alexa Fluor® 555 Conjugate) #8493 (red), and Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #3458 (blue pseudocolor).
    Cell Cycle Regulation Antibody Sampler Kit: Image 26 Expand Image
    Immunoprecipitation of p21 from human umbillical vein endothelial cells (HUVECs) using p21 Waf1/Cip1 (12D1) Rabbit mAb. Western blot detection was performed using the same antibody.
    Cell Cycle Regulation Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Cyclin D1 (92G2) Rabbit mAb in the presence of control peptide (left) or Cyclin D1 Blocking Peptide #1044 (right).
    Cell Cycle Regulation Antibody Sampler Kit: Image 28 Expand Image
    Confocal immunofluorescent analysis of MCF-7 cells using p27 Kip1 (D69C12) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
    Cell Cycle Regulation Antibody Sampler Kit: Image 29 Expand Image
    Flow cytometric analysis of Jurkat cells using CDK4 (D9G3E) Rabbit mAb (right) and Propidium Iodide (PI)/RNase Staining Solution #4087, compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Cell Cycle Regulation Antibody Sampler Kit: Image 30 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using p21 Waf1/Cip1 (12D1) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Cell Cycle Regulation Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded H1975 xenograft, using Cyclin D1 (92G2) Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded HeLa cells, transfected with SignalSilence® Control siRNA (Unconjugated) #6568 (left) or SignalSilence® p21 Waf1/Cip1 siRNA II #6558 (right), using p21 Waf1/Cip1 (12D1) Rabbit mAb.
    Cell Cycle Regulation Antibody Sampler Kit: Image 33 Expand Image
    Confocal immunofluorescent analysis of MCF7 cells using p21 Waf1/Cip1 (12D1) Rabbit mAb (red) and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Cell Cycle Regulation Antibody Sampler Kit: Image 34 Expand Image
    Flow cytometric analysis of Daudi cells using p21 Waf1/Cip1 (12D1) Rabbit mAb (right) and Propidium Iodide (PI)/RNase Staining Solution #4087, compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.