Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-cdc2 (Tyr15) Sandwich ELISA Kit #7176

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Kit Includes Volume Solution Color
Cdc2 (Y 15) Ab coatedmicrowells 96 tests
Cdc2 Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

CST's PathScan® Phospho-cdc2 (Tyr15) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-cdc2 (Tyr15) protein. A Phospho-cdc2 (Tyr15) Rabbit polyclonal Ab has been coated onto the microwells. After incubation with cell lysates, phospho-cdc2 (Tyr15) protein is captured by the coated antibody. Following extensive washing, cdc2 Mouse mAb is added to detect the captured phospho-cdc2 protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-cdc2 (Tyr15) protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-cdc2 (Tyr15) Sandwich ELISA Kit detects endogenous levels of phospho-cdc2 (Tyr15) protein. As shown in Figure 1, using the Phospho-cdc2 (Tyr15) ELISA Kit #7176, a significant induction of phospho-cdc2 (Tyr15) is detected in HeLa cells treated with Interleukin-4. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Sandwich ELISA

Sandwich ELISA

Figure 2: The relationship between protein concentration of lysates from lambda phosphatase and IL-4 treated HeLa cells and kit assay optical density reading. HeLa cells (80% confluent) were treated with human IL-4 for 10 minutes (100 ng/ml).

Sandwich ELISA

Sandwich ELISA

Figure 1: Treatment of HeLa cells with Interleukin-4 stimulates phosphorylation of cdc2 at Tyr15 as detected by PathScan® Phospho-cdc2 (Tyr15) Sandwich ELISA kit #7176, but does not affect the level of total cdc2 protein detected by cdc2 Antibody (#9116). OD 450 readings are shown in the top figure, while the corresponding Western blots using Phospho-cdc2 (Tyr15) Antibody #9111 (left panel) or cdc2 Antibody #9116 (right panel) is shown in the bottom figure.

Background

The entry of eukaryotic cells into mitosis is regulated by cdc2 kinase activation, a process controlled at several steps including cyclin binding and phosphorylation of cdc2 at Thr161 (1). However, the critical regulatory step in activating cdc2 during progression into mitosis appears to be dephosphorylation of cdc2 at Thr14 and Tyr15 (2). Phosphorylation at Thr14 and Tyr15, resulting in inhibition of cdc2, can be carried out by Wee1 and Myt1 protein kinases (3,4). The cdc25 phosphatase may be responsible for removal of phosphates at Thr14 and Tyr15 and subsequent activation of cdc2 (1,5).

  1. Atherton-Fessler, S. et al. (1994) Mol. Biol. Cell. 5, 989-1001.
  2. Norbury, C. et al. (1991) EMBO. J. 10, 3321-3329.
  3. McGowan, C.H. and Russell, P. (1993) EMBO J. 12, 75-85.
  4. Wells, N.J. et al. (1999) J. Cell. Sci. 112, 3361-3371.
  5. Hunter, T. (1995) Cell 80, 225-236.

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