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17498
Cell Cycle Phase Determination Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Cell Cycle Phase Determination Antibody Sampler Kit #17498

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Cell Cycle Phase Determination Antibody Sampler Kit: Image 1

Confocal immunofluorescent analysis of Hep G2 cells (left) and SK-MEL-28 cells (right) using Geminin (E5Q9S) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Cell Cycle Phase Determination Antibody Sampler Kit: Image 2

Confocal immunofluorescent analysis of HT-29 and HUVEC cells using CDT1 (D10F11) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Cell Cycle Phase Determination Antibody Sampler Kit: Image 3

Flow cytometric analysis of HCT 116 cells, wild-type (green, positive) or thymidine kinase 1 knockout (blue, negative), using Thymidine Kinase 1 (E2H7Z) Rabbit mAb (solid lines) or a 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) #4412 was used as a secondary antibody.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 4

HeLa cells were cultured and either left untreated (left panel) or treated with Nocodazole #2190 (100 ng/ml, 16 hr; right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin and Phospho-Histone H3 (Ser10) (D7N8E) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Promoter Primers #4471 and SimpleChIP® Human γ-Actin Promoter Primers #5037. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 5

Western blot analysis of extracts from various cell lines using Cyclin A2 (E1D9T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). In lane 6, HT-29 cells were treated with aphidicolin (10 µg/mL, 24 hr) to enrich cells at G1/S phase.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 6

Flow cytometric analysis of Jurkat cells using Cyclin B1 (D5C10) XP® Rabbit mAb and Propidium Iodide (PI)/RNase Staining Solution #4087 (DNA content). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 7

Western blot analysis of extracts from various cell lines using Cyclin E1 (D7T3U) Rabbit mAb.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 8

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

Cell Cycle Phase Determination Antibody Sampler Kit: Image 9

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 Phase Determination Antibody Sampler Kit: Image 10

Immunohistochemical analysis of paraffin-embedded 293T cell pellet (left, high-expressing) or SK-MEL-28 cell pellet (right, low-expressing) using Geminin (E5Q9S) XP® Rabbit mAb.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 11

Immunoprecipitation of CDT1 from HeLa cells using CDT1 (D10F11) Rabbit mAb. Western blot was performed using the same antibody. Lane 1 is 10% input.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 12

Confocal immunofluorescent analysis of HCT 116 cells, wild-type (left, positive) or thymidine kinase 1 knockout (right, negative), using Thymidine Kinase 1 (E2H7Z) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Cell Cycle Phase Determination Antibody Sampler Kit: Image 13

Flow cytometric analysis of Jurkat cells using Phospho-Histone H3 (Ser10) (D7N8E) 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 Phase Determination Antibody Sampler Kit: Image 14

Confocal immunofluorescent analysis of HT-29 cells using Cyclin B1 (D5C10) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Cell Cycle Phase Determination Antibody Sampler Kit: Image 15

Western blot analysis of extracts from HT-29 cells, untreated (-) or treated with aphidicolin (10 μg/ml; 24 hr; +) using Cyclin E1 (D7T3U) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Cell Cycle Phase Determination Antibody Sampler Kit: Image 16

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 Phase Determination Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using Geminin (E5Q9S) XP® Rabbit mAb.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 18

Western blot analysis of extracts from various cell lines using CDT1 (D10F11) Rabbit mAb.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 19

Immunoprecipitation of Thymidine Kinase 1 from HT-29 cell extracts treated with aphidicolin (10 μg/ml, 24 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Thymidine Kinase 1 (E2H7Z) Rabbit mAb. Western blot analysis was performed using Thymidine Kinase 1 (E2H7Z) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 20

Confocal immunofluorescent analysis of mitotic HeLa cells using Phospho-Histone H3 (Ser10) (D7N8E) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Cell Cycle Phase Determination Antibody Sampler Kit: Image 21

Western blot analysis of extracts from various cell lines using Cyclin B1 (D5C10) XP® Rabbit mAb.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 22

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 Phase Determination Antibody Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded human esophageal carcinoma using Geminin (E5Q9S) XP® Rabbit mAb.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 24

Western blot analysis of extracts from HT-29 cells, mock treated (-) or aphidicolin-treated (10 μg/ml, 24 hr; +) and HCT 116 cells, wild-type (-) or thymidine kinase 1 knockout (+), using Thymidine Kinase 1 (E2H7Z) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Cell Cycle Phase Determination Antibody Sampler Kit: Image 25

Western blot analysis of extracts from HeLa cells, either untreated or treated with Nocodazole #2190 (100 ng/ml, 16 hr), using Phospho-Histone H3 (Ser10) (D7N8E) XP® Rabbit mAb (upper) or Histone H3 (D1H2) Rabbit mAb #4499 (lower). Phospho-specificity of the antibody is shown by further treatment of the lysate with λ phosphatase.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 26

Western blot analysis of extracts from HT-29 cells, synchronized in S-phase by double thymidine block (2 nM, 16 hr) followed by release into fresh media for the indicated time, using Cyclin B1 (D5C10) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Cell Cycle Phase Determination Antibody Sampler Kit: Image 27

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

Cell Cycle Phase Determination Antibody Sampler Kit: Image 28

Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using Geminin (E5Q9S) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).

Cell Cycle Phase Determination Antibody Sampler Kit: Image 29

Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using Geminin (E5Q9S) XP® Rabbit mAb.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 30

Immunohistochemical analysis of paraffin-embedded human B-cell non-Hodgkin's lymphoma using Geminin (E5Q9S) XP® Rabbit mAb.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 31

Immunohistochemical analysis of paraffin-embedded human ovarian serous carcinoma using Geminin (E5Q9S) XP® Rabbit mAb.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 32

Immunoprecipitation of Geminin from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Geminin (E5Q9S) XP® Rabbit mAb. Western blot analysis was performed using

Geminin (E5Q9S) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.

Cell Cycle Phase Determination Antibody Sampler Kit: Image 33

Western bot analysis of extracts from various cell lines using Geminin (E5Q9S) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

To Purchase # 17498T
Product # Size Price
17498T
1 Kit  (8 x 20 µl) N/A

Product Description

The Cell Cycle Phase Determination Antibody Sampler Kit provides an economical means of detecting total proteins or post-translational modifications present in cells at various phases of the cell cycle. Geminin is degraded in G1 phase, while CDT1 is degraded in S, G2, and M phases. Thymidine Kinase 1 accumulates in G1 phase, peaks in S phase, and is degraded before cell division. Phospho-Histone H3 (Ser10) is present only in M phase, while Phospho-cdc2 (Tyr15) is absent in M phase. Cyclins A2, B1, and E1 peak at G2 phase, late G2/M phase, and late G1/early S phase, respectively. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Phospho-Histone H3 (Ser10) (D7N8E) XP® Rabbit mAb recognizes endogenous levels of histone H3 protein only when phosphorylated at Ser10. This antibody detects phosphorylation at Ser10 in the presence of acetylated or methylated Lys9, but not in the presence of phosphorylated Thr11. This antibody does not cross-react with histone H3 phosphorylated at Ser28.

Cyclin A2 (E1D9T) Rabbit mAb recognizes endogenous levels of total cyclin A2 protein.

Cyclin B1 (D5C10) XP® Rabbit mAb recognizes endogenous levels of total cyclin B1 protein. This antibody also detects a 100 kDa protein of unknown origin in some cell lines.

Cyclin E1 (D7T3U) Rabbit mAb recognizes endogenous levels of total cyclin E1 protein. Based on the sequence of the peptide antigen, this antibody is expected to detect all isoforms of cyclin E1. This antibody does not cross-react with cyclin E2.

Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb detects endogenous levels of cdc2 protein only when phosphorylated at tyrosine 15.

Geminin (E5Q9S) XP® Rabbit mAb recognizes endogenous levels of total geminin protein.

CDT1 (D10F11) Rabbit mAb recognizes endogenous levels of total CDT1 protein.

Thymidine Kinase 1 (E2H7Z) Rabbit mAb recognizes endogenous levels of total thymidine kinase 1 protein.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding phosphorylated Ser10 of human histone H3 protein, phosphorylated Tyr15 of human cdc2 protein, Val421 of human cyclin A2 protein, Pro209 of human thymidine kinase 1 protein, Gly351 of human cyclin E1 protein, Pro70 of human geminin protein, residues near the amino terminus of human CDT1 protein, and residues near the amino terminus of human cyclin B1 protein.

Background

The entry of eukaryotic cells into mitosis is regulated by cdc2/CDK1 kinase activation, a process controlled at several steps including cyclin B1 nuclear accumulation and binding, and phosphorylation of cdc2/CDK1 at Thr161 (1). At the end of mitosis, cyclin B1 is targeted for degradation by the anaphase-promoting complex (APC), allowing for cell cycle progression (2). A critical regulatory step in activating cdc2 during progression into mitosis is dephosphorylation of cdc2/CDK1 at Thr14 and Tyr15 (3).

Phosphorylation of Histone H3 at Ser10 is tightly correlated with chromosome condensation during both mitosis and meiosis (4).

Overcoming the G1/S checkpoint to commence DNA replication requires cyclin E, traversing the G2/M checkpoint to initiate mitosis requires cyclin B, and cyclin A is required for both S-phase and M-phase (5). Cyclin A availability is apparently the rate-limiting step for entry into mitosis, and cyclin A is required for completion of prophase (6).

Thymidine kinases play a critical role in generating the DNA synthetic precursor deoxythymidine triphosphate (dTTP). Cytoplasmic thymidine kinase 1 (TK1) expression and activity are regulated in a cell cycle-dependent manner, accumulating during G1-phase to peak levels in S-phase before being degraded prior to cell division (7).

The initiation of S phase begins with the formation of the pre-replication complex (pre-RC) in late mitosis/early G1 phase. CDT1 and cdc6 bind to the origin of DNA replication, which allows binding of the MCM2-7 complex. In order to ensure that replication occurs only once per cell cycle, geminin inhibits and destabilizes CDT1 during the S, G2 and M phases. At the metaphase/anaphase transition, geminin is degraded by the anaphase-promoting complex (APC) allowing for the formation of new pre-RC (8).

  1. Atherton-Fessler, S. et al. (1994) Mol Biol Cell 5, 989-1001.
  2. Gong, D. and Ferrell, J.E. (2010) Mol Biol Cell 21, 3149-61.
  3. Norbury, C. et al. (1991) EMBO J 10, 3321-9.
  4. Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60.
  5. Pagano, M. et al. (1992) EMBO J 11, 961-71.
  6. Furuno, N. et al. (1999) J. Cell. Biol. 147, 295-306.
  7. Munch-Petersen, B. (2010) Nucleosides Nucleotides Nucleic Acids 29, 363-9.
  8. Caillat, C. and Perrakis, A. (2012) Subcell Biochem 62, 71-87.

Pathways & Proteins

Explore pathways + proteins related to this product.

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'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.
XP is a registered trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 5,675,063.
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