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).Learn more about how we get our images
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.Learn more about how we get our images
|Product Includes||Volume||Solution Color|
|Phospho-Cdc2 (Tyr15) Rabbit mAb Coated Microwells||96 tests|
|Cdc2 Detection Mouse mAb||11 ml||Green|
|Anti-mouse IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.
Add 100 µl of STOP solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.
posted June 2005
revised November 2013
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.
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.
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PathScan is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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