Revision 1

#17498Store at -20C

1 Kit

(8 x 20 microliters)

Cell Signaling Technology

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

Support: 877-678-TECH (8324)

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3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Geminin (E5Q9S) XP® Rabbit mAb 52508 20 µl 25 kDa Rabbit IgG
CDT1 (D10F11) Rabbit mAb 8064 20 µl 65 kDa Rabbit IgG
Thymidine Kinase 1 (E2H7Z) Rabbit mAb 28755 20 µl 26 kDa Rabbit IgG
Phospho-Histone H3 (Ser10) (D7N8E) XP® Rabbit mAb 53348 20 µl 17 kDa Rabbit IgG
Cyclin A2 (E1D9T) Rabbit mAb 91500 20 µl 55 kDa Rabbit IgG
Cyclin B1 (D5C10) XP® Rabbit mAb 12231 20 µl 55 kDa Rabbit IgG
Cyclin E1 (D7T3U) Rabbit mAb 20808 20 µl 48 kDa Rabbit IgG
Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb 4539 20 µl 34 kDa Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

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

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.

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

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.

Background References

    Trademarks and Patents

    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.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #17498

    Cell Cycle Phase Determination Antibody Sampler Kit

    Cell Cycle Phase Determination Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from HeLa cells using Cyclin B1 (D5C10) XP® Rabbit mAb #12231. The virtual lane view (left) shows the 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 Phase Determination Antibody Sampler Kit: Image 2 Expand Image
    Simple WesternTM analysis of lysates (0.1 mg/mL) from Jurkat cells using Cyclin A2 (E1D9T) Rabbit mAb #91500. The virtual lane view (left) shows the 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 JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Cell Cycle Phase Determination Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from various cell lines using Cyclin B1 (D5C10) XP® Rabbit mAb.
    Cell Cycle Phase Determination Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using Cyclin E1 (D7T3U) Rabbit mAb.
    Cell Cycle Phase Determination Antibody Sampler Kit: Image 5 Expand Image
    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 6 Expand Image
    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 7 Expand Image
    Western bot analysis of extracts from various cell lines using Geminin (E5Q9S) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Cell Cycle Phase Determination Antibody Sampler Kit: Image 8 Expand Image
    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 9 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 Phase Determination Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using CDT1 (D10F11) Rabbit mAb.
    Cell Cycle Phase Determination Antibody Sampler Kit: Image 11 Expand Image
    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 12 Expand Image
    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 13 Expand Image
    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 14 Expand Image
    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 15 Expand Image
    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 16 Expand Image
    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 17 Expand Image
    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 18 Expand Image
    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 19 Expand Image
    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 20 Expand Image
    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 21 Expand Image
    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 22 Expand Image
    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 23 Expand Image
    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 24 Expand Image
    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 25 Expand Image
    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 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using Geminin (E5Q9S) XP® Rabbit mAb.
    Cell Cycle Phase Determination Antibody Sampler Kit: Image 27 Expand Image
    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 28 Expand Image
    Flow cytometric analysis of Jurkat cells using Cyclin B1 (D5C10) XP® 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 Phase Determination Antibody Sampler Kit: Image 29 Expand Image
    Immunohistochemical analysis of paraffin-embedded human esophageal carcinoma using Geminin (E5Q9S) XP® Rabbit mAb.
    Cell Cycle Phase Determination Antibody Sampler Kit: Image 30 Expand Image
    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 31 Expand Image
    Flow cytometric analysis of Jurkat cells using Phospho-cdc2 (Tyr15) (10A11) Rabbit mAb (right) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left), co-stained with Propidium Iodide (PI)/RNase Staining Solution #4087. 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 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using Geminin (E5Q9S) XP® Rabbit mAb.
    Cell Cycle Phase Determination Antibody Sampler Kit: Image 33 Expand Image
    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 34 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ovarian serous carcinoma using Geminin (E5Q9S) XP® Rabbit mAb.
    Cell Cycle Phase Determination Antibody Sampler Kit: Image 35 Expand Image
    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).
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.