PTEN (Phospho-Ser380/Thr382/Thr383) Cell-Based ELISA Kit

Product Name:	PTEN (Phospho-Ser380/Thr382/Thr383) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01601
ELISA Type:	Cell-Based
Target:	Phospho-PTEN (Ser380/Thr382/Thr383)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The PTEN (Phospho-Ser380/Thr382/Thr383) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PTEN protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PTEN in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PTEN phosphorylation.

Qualitative determination of PTEN (Phospho-Ser380/Thr382/Thr383) concentration is achieved by an indirect ELISA format. In essence, PTEN (Phospho-Ser380/Thr382/Thr383) is captured by PTEN (Phospho-Ser380/Thr382/Thr383)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 5728, UniProt ID: P60484, OMIM: 137800/153480/158350/176807/176920/275355/276950/601728/605309/608089/612242, Unigene: Hs.500466
Gene Symbol:	PTEN
Sub Type:	Phospho

UniProt Protein Function:	PTEN: a phosphoinositide 3-phosphatase (PIP3) and a tumor suppressor implicated in a wide variety of human cancers. Dephosphorylates inositol phospholipids generated by the activation of the phosphoinositide 3 kinase (PI3K). A major negative regulator of the PI3K/Akt signaling pathway. Possesses a C-terminal regulatory domain that contains three phosphorylation sites which may regulate its stability.
UniProt Protein Details:	Protein type:Carbohydrate Metabolism - inositol phosphate; EC 3.1.3.48; Phosphatase, lipid; Nuclear receptor co-regulator; Motility/polarity/chemotaxis; EC 3.1.3.16; EC 3.1.3.67; Tumor suppressor Chromosomal Location of Human Ortholog: 10q23.3 Cellular Component: nucleoplasm; postsynaptic membrane; PML body; internal side of plasma membrane; neuron projection; cell projection; mitochondrion; cytoplasm; plasma membrane; extracellular region; dendritic spine; cytosol; nucleus Molecular Function:phosphatidylinositol-3-phosphatase activity; phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase activity; inositol-1,3,4,5-tetrakisphosphate 3-phosphatase activity; platelet-derived growth factor receptor binding; magnesium ion binding; protein tyrosine phosphatase activity; protein serine/threonine phosphatase activity; protein kinase binding; PDZ domain binding; protein binding; enzyme binding; phosphatidylinositol-3,4-bisphosphate 3-phosphatase activity; protein tyrosine/serine/threonine phosphatase activity; phosphoprotein phosphatase activity; lipid binding Biological Process: central nervous system development; male mating behavior; nerve growth factor receptor signaling pathway; prepulse inhibition; response to arsenic; regulation of cellular component size; heart development; anaphase-promoting complex activation during mitotic cell cycle; Wnt receptor signaling pathway through beta-catenin; T cell receptor signaling pathway; regulation of B cell apoptosis; myelin maintenance in the central nervous system; response to glucose stimulus; negative regulation of axonogenesis; response to drug; rhythmic synaptic transmission; fibroblast growth factor receptor signaling pathway; dentate gyrus development; negative regulation of myelination; social behavior; forebrain morphogenesis; negative regulation of organ growth; response to ethanol; response to zinc ion; inositol phosphate dephosphorylation; phosphatidylinositol biosynthetic process; endothelial cell migration; positive regulation of transcription factor activity; multicellular organismal response to stress; negative regulation of apoptosis; negative regulation of phagocytosis; synapse maturation; inositol phosphate metabolic process; apoptosis; regulation of protein stability; locomotory behavior; negative regulation of cell size; platelet-derived growth factor receptor signaling pathway; protein amino acid dephosphorylation; regulation of myeloid cell apoptosis; response to estradiol stimulus; negative regulation of cell proliferation; locomotor rhythm; learning and/or memory; synaptogenesis; negative regulation of protein amino acid phosphorylation; phospholipid metabolic process; positive regulation of cell proliferation; brain morphogenesis; protein kinase B signaling cascade; nerve-nerve synaptic transmission; angiogenesis; negative regulation of cell migration; response to nutrient; negative regulation of epithelial cell proliferation; aging; cardiac muscle development; epidermal growth factor receptor signaling pathway; cell migration; phosphoinositide-mediated signaling; negative regulation of protein kinase B signaling cascade; protein stabilization; central nervous system neuron axonogenesis; maternal behavior; negative regulation of focal adhesion formation; response to ATP; memory; cell proliferation; G1/S-specific negative regulation of cyclin-dependent protein kinase activity; innate immune response; phosphoinositide dephosphorylation; regulation of cyclin-dependent protein kinase activity; negative regulation of phosphoinositide 3-kinase cascade Disease: Meningioma, Familial, Susceptibility To; Thyroid Carcinoma, Follicular; Melanoma, Cutaneous Malignant, Susceptibility To, 1; Bannayan-riley-ruvalcaba Syndrome; Cowden Syndrome 1; Squamous Cell Carcinoma, Head And Neck; Prostate Cancer; Glioma Susceptibility 2; Vacterl Association With Hydrocephalus; Endometrial Cancer; Macrocephaly/autism Syndrome
NCBI Summary:	This gene was identified as a tumor suppressor that is mutated in a large number of cancers at high frequency. The protein encoded this gene is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It contains a tensin like domain as well as a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating AKT/PKB signaling pathway. [provided by RefSeq, Jul 2008]
UniProt Code:	P60484
NCBI GenInfo Identifier:	42560209
NCBI Gene ID:	5728
NCBI Accession:	P60484.1
UniProt Secondary Accession:	P60484,O00633, O02679, Q6ICT7, B2R904, F2YHV0,
UniProt Related Accession:	P60484
Molecular Weight:	19,796 Da
NCBI Full Name:	Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN
NCBI Synonym Full Names:	phosphatase and tensin homolog
NCBI Official Symbol:	PTEN
NCBI Official Synonym Symbols:	BZS; DEC; CWS1; GLM2; MHAM; TEP1; MMAC1; PTEN1; 10q23del
NCBI Protein Information:	phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN; mitochondrial PTENalpha; phosphatase and tensin-like protein; mutated in multiple advanced cancers 1; mitochondrial phosphatase and tensin protein alpha; MMAC1 phosphatase and tensin homolog deleted on chromosome 10; phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN
UniProt Protein Name:	Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN
UniProt Synonym Protein Names:	Mutated in multiple advanced cancers 1; Phosphatase and tensin homolog
Protein Family:	Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase
UniProt Gene Name:	PTEN
UniProt Entry Name:	PTEN_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.	Incubate the cells for overnight at 37 °C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-PTEN (Phospho-Ser380/Thr382/Thr383) Antibody, Anti-PTEN Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

Tigatuzumab: Advancing Cancer Research with Targeted Therapies

Enavatuzumab: Revolutionizing Cancer Research Through Novel Therapeutics

Alemtuzumab: Mechanism, Applications, and Biosimilar Advancements

PTEN (Phospho-Ser380/Thr382/Thr383) Colorimetric Cell-Based ELISA Kit (CBCAB01601)

Description

PTEN (Phospho-Ser380/Thr382/Thr383)Colorimetric Cell-Based ELISA Kit

Overview

Product Description

Additional Information

Kit Components

Protocol