PTP1B (Phospho-Ser50)Colorimetric Cell-Based ELISA Kit (CBCAB01381)

Product Name:	PTP1B (Phospho-Ser50)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01381
ELISA Type:	Cell-Based
Target:	PTP1B (Phospho-Ser50)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The PTP1B (Phospho-Ser50) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PTP1B protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PTP1B in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PTP1B phosphorylation.

Qualitative determination of PTP1B (Phospho-Ser50) concentration is achieved by an indirect ELISA format. In essence, PTP1B (Phospho-Ser50) is captured by PTP1B (Phospho-Ser50)-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: 5770, UniProt ID: P18031, OMIM: 176885, Unigene: Hs.417549
Gene Symbol:	PTPN1
Sub Type:	Phospho

UniProt Protein Function:	PTP1B: a non-receptor phospho-tyrosine protein phosphatase which acts as a regulator of endoplasmic reticulum unfolded protein response. Mediates dephosphorylation and inactivation of PERK. May play an important role in CKII- and p60c-src-induced signal transduction cascades. May regulate the EFNA5-EPHA3 signaling pathway which modulates cell reorganization and cell-cell repulsion. Negatively regulates insulin signaling by dephosphorylating the phosphotyrosine residues of insulin receptor. Also reported to dephosphorylate integrin, epidermal growth factor receptor, JAK2 and TYK2, regulating cell growth control and the cellular response to interferon. May regulate the hepatocyte growth factor receptor signaling pathway through dephosphorylation of MET. PTP1B knockout mice show resistance to dietary weight gain and enhanced insulin sensitivity, suggesting a possible role in treatment of obesity as well as type 2 diabetes.
UniProt Protein Details:	Protein type:EC 3.1.3.48; Protein phosphatase, tyrosine (non-receptor); Motility/polarity/chemotaxis; Phosphatase Chromosomal Location of Human Ortholog: 20q13.1-q13.2 Cellular Component: endoplasmic reticulum; early endosome; plasma membrane; cytoplasmic vesicle; cytosol Molecular Function:protein binding; enzyme binding; ephrin receptor binding; zinc ion binding; protein tyrosine phosphatase activity; receptor tyrosine kinase binding; protein kinase binding; insulin receptor binding Biological Process: platelet activation; unfolded protein response, activation of signaling protein activity; negative regulation of vascular endothelial growth factor receptor signaling pathway; unfolded protein response; cytokine and chemokine mediated signaling pathway; actin cytoskeleton reorganization; regulation of endocytosis; insulin receptor signaling pathway; regulation of signal transduction; negative regulation of insulin receptor signaling pathway; blood coagulation Disease: Diabetes Mellitus, Noninsulin-dependent
NCBI Summary:	The protein encoded by this gene is the founding member of the protein tyrosine phosphatase (PTP) family, which was isolated and identified based on its enzymatic activity and amino acid sequence. PTPs catalyze the hydrolysis of the phosphate monoesters specifically on tyrosine residues. Members of the PTP family share a highly conserved catalytic motif, which is essential for the catalytic activity. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. This PTP has been shown to act as a negative regulator of insulin signaling by dephosphorylating the phosphotryosine residues of insulin receptor kinase. This PTP was also reported to dephosphorylate epidermal growth factor receptor kinase, as well as JAK2 and TYK2 kinases, which implicated the role of this PTP in cell growth control, and cell response to interferon stimulation. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2013]
UniProt Code:	P18031
NCBI GenInfo Identifier:	131467
NCBI Gene ID:	5770
NCBI Accession:	P18031.1
UniProt Secondary Accession:	P18031,Q5TGD8, Q9BQV9, Q9NQQ4,
UniProt Related Accession:	P18031
Molecular Weight:	49,967 Da
NCBI Full Name:	Tyrosine-protein phosphatase non-receptor type 1
NCBI Synonym Full Names:	protein tyrosine phosphatase, non-receptor type 1
NCBI Official Symbol:	PTPN1
NCBI Official Synonym Symbols:	PTP1B
NCBI Protein Information:	tyrosine-protein phosphatase non-receptor type 1; protein-tyrosine phosphatase 1B; protein tyrosine phosphatase, placental
UniProt Protein Name:	Tyrosine-protein phosphatase non-receptor type 1
UniProt Synonym Protein Names:	Protein-tyrosine phosphatase 1B; PTP-1B
Protein Family:	Tyrosine-protein phosphatase non-receptor
UniProt Gene Name:	PTPN1
UniProt Entry Name:	PTN1_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-PTP1B (Phospho-Ser50) Antibody, Anti-PTP1B 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.)