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9917
Cell Cycle/Checkpoint Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Cell Cycle/Checkpoint Antibody Sampler Kit #9917

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Cell Cycle/Checkpoint Antibody Sampler Kit: Image 1

Flow cytometric analysis of Jurkat cells, using Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb versus propidium iodide (DNA content).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 2

Flow cytometric analysis of HeLa cells, untreated (blue) and UV-treated (100 mJ/cm2, 1 hr recovery; green), using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 3

Flow cytometric analysis of HeLa cells, untreated (blue) or treated with UV (100mJ/cm2, 2 hr recovery; green) using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) was used as a secondary antibody.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 4

Flow cytometric analysis of Jurkat cells using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb 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/Checkpoint Antibody Sampler Kit: Image 5

Western blot analysis of extracts from human fibroblasts synchronized by serum deprivation, using Phospho-Rb (Ser795) Antibody. Cells were synchronized for 24 hours, then released by addition of serum and harvested at the times indicated. Cell cycle progression was verified by cyclin analysis and FACS. (Provided by John Boylan, Dupont/Merck, Delaware.)

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 6

Flow cytometric analysis of HT-29 cells, untreated (blue) or UV-treated (green), using Phospho-p53 (Ser15) (16G8) Mouse mAb compared to a nonspecific negative control antibody (red).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 7

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/Checkpoint Antibody Sampler Kit: Image 8

Confocal immunofluorescent analysis of asynchronous HeLa cells labeled with Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb (green) and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (red).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 9

Confocal immunofluorescent analysis of C2C12 cells, untreated (left) or UV-treated (right), using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 10

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 11

Confocal immunofluorescent analysis of MCF7 (left) and BT-549 (right) cells, untreated (upper) or λ phosphatase-treated (lower) using Phospho-Rb (Ser807/Ser811) (D20B12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 12

Western blot analysis of Rb Control Protein #9303, using Phospho-Rb (Ser795) Antibody (upper) or Rb (4H1) mAb #9309 (lower).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 13

Confocal immunofluorescent analysis of HT-29 cells, untreated (left) or UV-treated (right), using Phospho-p53 (Ser15) (16G8) Mouse mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 14

Western blot analysis of extracts from C6 cells, untreated (-) or treated with Nocodazole (0.1 μg/ml, 18 hr; +), using Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb (upper) or cdc2 Antibody #77055 (lower).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 15

Western blot analysis of extracts from HeLa, COS, NIH/3T3 and C6 cells, untreated or UV-treated, using Phospho-Chk1 (Ser345) (133D30) Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 16

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 18

Western blot analysis of extracts from HT29 cells, untreated or UV-treated (100 mJ/cm2, 1 hr), using Phospho-p53 (Ser15) (16G8) Mouse mAb (upper) or p53 (DO-7) Mouse mAb #48818 (lower).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 19

Western blot analysis of extracts from HeLa cells, untreated or hydroxyurea treated for 20 hours, using Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 20

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 21

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded HT-29 cell pellets, control (left) or UV-treated (right), using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded mouse spleen using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 24

Immunoprecipitation of phospho-chk2 from UV-treated HT29 cells using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb followed by western blot using the same antibody.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 25

Immunohistochemical analysis of paraffin-embedded human ovarian serous adenocarcinoma using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 26

Western blot analysis of extracts from HeLa cells, untreated or UV-treated, using Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 27

Immunoprecipitation of phospho-Rb (Ser807/811) from Cos cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 28

Western blot analysis of extracts from MCF7 cells, untreated (-) or treated with calf intestinal phosphatase (CIP) and λ phosphatase (+), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (upper) or Rb (4H1) Mouse mAb #9309 (lower).

Cell Cycle/Checkpoint Antibody Sampler Kit: Image 29

Western blot analysis of extracts from WI-38 cells, serum-starved for 3 days (-) or serum-starved for 3 days followed by treatment with 10% serum for 2 days (+), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

To Purchase # 9917T
Product # Size Price
9917T
1 Kit  (6 x 20 µl) $ 445

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb 4539 20 µl
  • WB
  • IP
  • IF
  • F
H M R Mk 34 Rabbit 
Phospho-Chk1 (Ser345) (133D3) Rabbit mAb 2348 20 µl
  • WB
  • IF
  • F
H M R Mk 56 Rabbit IgG
Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb 2197 20 µl
  • WB
  • IP
  • IHC
  • F
H 62 Rabbit IgG
Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb 8516 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk 110 Rabbit IgG
Phospho-Rb (Ser795) Antibody 9301 20 µl
  • WB
  • IP
H R Mk 110 Rabbit 
Phospho-p53 (Ser15) (16G8) Mouse mAb 9286 20 µl
  • WB
  • IF
  • F
H 53 Mouse IgG1
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Product Description

The Cell Cycle/Checkpoint Antibody Sampler Kit provides a fast and economical means of evaluating multiple proteins involved in the cell cyle and checkpoint control. The kit contains enough primary and secondary antibody to perform four Western blot experiments.

Specificity / Sensitivity

Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb detects endogenous levels of cdc2 protein only when phosphorylated at tyrosine 15. Based on sequence similarity, the antibody may cross-react with CDK2 and CDK3. Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb detects endogenous levels of Chk2 only when phosphorylated at Thr68. Phospho-Chk1 (Ser345) Antibody detects Chk1 only when phosphorylated at Ser345 and does not cross-react with other proteins. Phospho-Rb (Ser795) Antibody detects Rb only when phosphorylated at Ser795 and does not cross-react with Rb phosphorylated at other sites. Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb recognizes endogenous levels of Rb protein only when phosphorylated at Ser807, Ser811, or at both sites. This antibody does not cross-react with Rb phosphorylated at Ser608. Phospho-p53 (Ser15) (16G8) Mouse mAb detects endogenous levels of p53 only when phosphorylated at Ser15. The antibody does not cross-react with p53 phosphorylated at other sites.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser795 of human Rb. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser807/811 of human Rb protein, residues surrounding Ser345 of human Chk1, residues surrounding Ser15 of human p53, residues surrounding Tyr15 of human cdc2, and residues surrounding Thr68 of human Chk2.

Background

The cell division cycle demands accuracy to avoid the accumulation of genetic damage. This process is controlled by molecular circuits called "checkpoints" that are common to all eukaryotic cells (1). Checkpoints monitor DNA integrity and cell growth prior to replication and division at the G1/S and G2/M transitions, respectively. The cdc2-cyclin B kinase is pivotal in regulating the G2/M transition (2,3). Cdc2 is phosphorylated at Thr14 and Tyr15 during G2-phase by the kinases Wee1 and Myt1, rendering it inactive. The tumor suppressor protein retinoblastoma (Rb) controls progression through the late G1 restriction point (R) and is a major regulator of the G1/S transition (4). During early and mid G1-phase, Rb binds to and represses the transcription factor E2F (5). The phosphorylation of Rb late in G1-phase by CDKs induces Rb to dissociate from E2F, permitting the transcription of S-phase-promoting genes. In vitro, Rb can be phosphorylated at multiple sites by cdc2, cdk2, and cdk4/6 (6-8). DNA damage triggers both the G2/M and the G1/S checkpoints. DNA damage activates the DNA-PK/ATM/ATR kinases, which phosphorylate Chk at Ser345 (9), Chk2 at Thr68 (10) and p53 (11). The Chk kinases inactivate cdc25 via phosphorylation at Ser216, blocking the activation of cdc2.

  1. Nurse, P. (1997) Cell 91, 865-7.
  2. Norbury, C. and Nurse, P. (1992) Annu Rev Biochem 61, 441-70.
  3. Watanabe, N. et al. (1995) EMBO J. 14, 1878-1891.
  4. Sherr, C.J. (1996) Science 274, 1672-7.
  5. Dyson, N. (1998) Genes Dev 12, 2245-62.
  6. Kitagawa, M. et al. (1996) EMBO J 15, 7060-9.
  7. Lundberg, A.S. and Weinberg, R.A. (1998) Mol Cell Biol 18, 753-61.
  8. Harbour, J.W. et al. (1999) Cell 98, 859-69.
  9. Zhao, H. and Piwnica-Worms, H. (2001) Mol. Cell. Biol. 21, 4129-4139.
  10. Matsuoka, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10389-10394.
  11. Tibbetts, R.S. et al. (1999) Genes Dev 13, 152-7.

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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's 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's 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.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.