PYK2 (Phospho-Tyr580) Colorimetric Cell-Based ELISA Kit

Product Name:	PYK2 (Phospho-Tyr580) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01249
ELISA Type:	Cell-Based
Target:	PYK2 (Phospho-Tyr580)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The PYK2 (Phospho-Tyr580) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PYK2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PYK2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PYK2 phosphorylation.

Qualitative determination of PYK2 (Phospho-Tyr580) concentration is achieved by an indirect ELISA format. In essence, PYK2 (Phospho-Tyr580) is captured by PYK2 (Phospho-Tyr580)-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: 2185, UniProt ID: Q14289, OMIM: 601212, Unigene: Hs.491322
Gene Symbol:	PTK2B
Sub Type:	Phospho

UniProt Protein Function:	Pyk2: a nonreceptor tyrosine kinase of the Fak family. Predominantly expressed in the cells derived from hematopoietic lineages and in the central nervous system. Pyk2 is one of the signaling mediators for G-protein-coupled receptors. Involved in calcium induced regulation of ion channel and activation of the map kinase signaling pathway. Interacts with the SH2 domain of Grb2. May phosphorylate the voltage-gated potassium channel protein Kv1.2. Its activation is highly correlated with the stimulation of c-Jun N-terminal kinase activity. It plays an important role in cell motility such as spreading and migration. Two alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:Nuclear receptor co-regulator; Protein kinase, TK; Kinase, protein; EC 2.7.10.2; Protein kinase, tyrosine (non-receptor); TK group; Fak family Chromosomal Location of Human Ortholog: 8p21.1 Cellular Component: axon; cell cortex; cell soma; cytoplasm; cytoskeleton; cytosol; dendrite; extrinsic to internal side of plasma membrane; focal adhesion; growth cone; lamellipodium; lipid raft; N-methyl-D-aspartate selective glutamate receptor complex; nucleoplasm; nucleus; perinuclear region of cytoplasm; postsynaptic density Molecular Function:3-phosphoinositide-dependent protein kinase binding; ATP binding; calmodulin-dependent protein kinase activity; N-methyl-D-aspartate selective glutamate receptor activity; non-membrane spanning protein tyrosine kinase activity; protein binding; protein complex binding; protein-tyrosine kinase activity; receptor binding; signal transducer activity Biological Process: activation of JAK protein; adaptive immune response; angiogenesis; apoptosis; blood vessel endothelial cell migration; bone resorption; cell surface receptor linked signal transduction; cellular defense response; elevation of cytosolic calcium ion concentration; epidermal growth factor receptor signaling pathway; focal adhesion formation; glial cell proliferation; innate immune response; integrin-mediated signaling pathway; ionotropic glutamate receptor signaling pathway; MAPKKK cascade; marginal zone B cell differentiation; negative regulation of apoptosis; negative regulation of bone mineralization; negative regulation of cell proliferation; negative regulation of myeloid cell differentiation; negative regulation of neuron apoptosis; negative regulation of potassium ion transport; neurite development; oocyte maturation; peptidyl-tyrosine phosphorylation; positive regulation of actin filament polymerization; positive regulation of angiogenesis; positive regulation of cell growth; positive regulation of cell migration; positive regulation of cell proliferation; positive regulation of cell-matrix adhesion; positive regulation of JNK activity; positive regulation of JNK cascade; positive regulation of nitric oxide biosynthetic process; positive regulation of nitric-oxide synthase activity; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of phosphoinositide 3-kinase activity; positive regulation of protein kinase activity; positive regulation of synaptic transmission, glutamatergic; positive regulation of translation; protein amino acid autophosphorylation; protein amino acid phosphorylation; protein complex assembly; regulation of calcium-mediated signaling; regulation of cell adhesion; regulation of cell shape; regulation of cGMP biosynthetic process; regulation of inositol trisphosphate biosynthetic process; regulation of release of sequestered calcium ion into cytosol; response to calcium ion; response to cAMP; response to cocaine; response to drug; response to ethanol; response to glucose stimulus; response to hormone stimulus; response to hydrogen peroxide; response to hypoxia; response to lithium ion; response to mechanical stimulus; response to osmotic stress; response to stress; signal complex assembly; signal transduction; sprouting angiogenesis; stress fiber formation; tumor necrosis factor-mediated signaling pathway; vascular endothelial growth factor receptor signaling pathway
NCBI Summary:	This gene encodes a cytoplasmic protein tyrosine kinase which is involved in calcium-induced regulation of ion channels and activation of the map kinase signaling pathway. The encoded protein may represent an important signaling intermediate between neuropeptide-activated receptors or neurotransmitters that increase calcium flux and the downstream signals that regulate neuronal activity. The encoded protein undergoes rapid tyrosine phosphorylation and activation in response to increases in the intracellular calcium concentration, nicotinic acetylcholine receptor activation, membrane depolarization, or protein kinase C activation. This protein has been shown to bind CRK-associated substrate, nephrocystin, GTPase regulator associated with FAK, and the SH2 domain of GRB2. The encoded protein is a member of the FAK subfamily of protein tyrosine kinases but lacks significant sequence similarity to kinases from other subfamilies. Four transcript variants encoding two different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	Q14289
NCBI GenInfo Identifier:	3183003
NCBI Gene ID:	2185
NCBI Accession:	Q14289.2
UniProt Secondary Accession:	Q14289,Q13475, Q14290, Q16709, Q6PID4, D3DST0,
UniProt Related Accession:	Q14289
Molecular Weight:	111,183 Da
NCBI Full Name:	Protein-tyrosine kinase 2-beta
NCBI Synonym Full Names:	protein tyrosine kinase 2 beta
NCBI Official Symbol:	PTK2B
NCBI Official Synonym Symbols:	PKB; PTK; CAKB; FAK2; PYK2; CADTK; FADK2; RAFTK
NCBI Protein Information:	protein-tyrosine kinase 2-beta
UniProt Protein Name:	Protein-tyrosine kinase 2-beta
UniProt Synonym Protein Names:	Calcium-dependent tyrosine kinase; CADTK; Calcium-regulated non-receptor proline-rich tyrosine kinase; Cell adhesion kinase beta; CAK-beta; CAKB; Focal adhesion kinase 2; FADK 2; Proline-rich tyrosine kinase 2; Related adhesion focal tyrosine kinase; RAFTK
UniProt Gene Name:	PTK2B
UniProt Entry Name:	FAK2_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-PYK2 (Phospho-Tyr580) Antibody, Anti-PYK2 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.)

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신경면역학: CNS의 면역체계

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PYK2 (Phospho-Tyr580) Colorimetric Cell-Based ELISA Kit

Description