Figure 1. Phosphorylation of PTEN at Ser380 detected by PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit #7285. PTEN in HeLa cells is constitutively phosphorylated at Ser380. Treatment of HeLa cell lysates with λ phosphatase (4000 U/mL for 60 minutes at 37ºC) abolishes the phosphorylation of PTEN at Ser380 as shown using PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit and by Western analysis. The level of total PTEN detected by Western analysis remains unchanged. Absorbance450 nm is shown in the top figure while the corresponding Western blots using Phospho-PTEN (Ser380) Antibody #9551 (right panel) or PTEN Antibody #9552 (left panel) are shown in the bottom figure.
Figure 2. The relationship between the protein concentration of untreated and λ phosphatase-treated HeLa cell lysates (see the legend of Figure 1) and the absorbance at 450 nm is shown.
|REACTIVITY||H M Mk|
|Product Includes||Volume||Solution Color|
|PTEN Mouse mAb Coated Microwells||96 tests|
|Phospho-PTEN (Ser380) Rabbit Detection Antibody||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||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|
CST's PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-PTEN (Ser380) protein. A PTEN Mouse mAb has been coated onto the microwells. After incubation with cell lysates, PTEN protein (phospho and non-phospho) is captured by the coated antibody. Following extensive washing, Phospho-PTEN (Ser380) Antibody is added to detect the captured phospho-PTEN (Ser380) protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of phospho-PTEN (Ser380) protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-PTEN (Ser380) Sandwich ELISA Kit detects endogenous levels of phospho-PTEN (Ser380) protein. As shown in Figure 1 using the Phospho-PTEN (Ser380) Sandwich ELISA Kit #7285 a high level of phospho-PTEN (Ser380) is detected in HeLa cells. These levels are significantly reduced in HeLa cell lysates treated with λ phosphatase. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Human, Mouse, Monkey
PTEN (phosphatase and tensin homologue deleted on chromosome ten), also referred to as MMAC (mutated in multiple advanced cancers) phosphatase, is a tumor suppressor implicated in a wide variety of human cancers (1). PTEN encodes a 403 amino acid polypeptide originally described as a dual-specificity protein phosphatase (2). The main substrates of PTEN are inositol phospholipids generated by the activation of the phosphoinositide 3-kinase (PI3K) (3). PTEN is a major negative regulator of the PI3K/Akt signaling pathway (1,4,5). PTEN possesses a carboxy-terminal, noncatalytic regulatory domain with three phosphorylation sites (Ser380, Thr382, and Thr383) that regulate PTEN stability and may affect its biological activity (6,7). PTEN regulates p53 protein levels and activity (8) and is involved in G protein-coupled signaling during chemotaxis (9,10).
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