Kit (Phospho-Tyr721) Colorimetric Cell-Based ELISA Kit

Product Name:	Kit (Phospho-Tyr721) Colorimetric Cell-Based ELISA
Product Code:	CBCAB00472
ELISA Type:	Cell-Based
Target:	Kit (Phospho-Tyr721)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Kit (Phospho-Tyr721) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Kit protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Kit in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Kit phosphorylation.

Qualitative determination of Kit (Phospho-Tyr721) concentration is achieved by an indirect ELISA format. In essence, Kit (Phospho-Tyr721) is captured by Kit (Phospho-Tyr721)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 3815, UniProt ID: P10721, OMIM: 164920/172800/273300/606764, Unigene: Hs.479754
Gene Symbol:	KIT
Sub Type:	Phospho

UniProt Protein Function:	Kit: a receptor tyrosine kinase and a member of the subfamily that includes PDGF, CSF-1 and FLT-3/flk-2 receptors. Receptor for stem cell factor. Plays a critical role in hematopoietic stem cell, mast cell, melanocyte and germ cell development. Ligand binding induces autophosphorylation, dimerization and activation, leading to the recruitment and phosphorylation of downstream SH2-containing signaling components including PLC-gamma, PI3 kinase p85, SHP2 and CrkL, linking c-Kit to various cell signaling pathways. Molecular lesions that impair the kinase activity of c-Kit are associated with a variety of developmental disorders, while mutations that constitutively activate c-Kit can lead to hyperplasia and tumorigenesis. Activating mutations cause >90% of gastrointestinal stromal tumors (GIST); successfully treated with inhibitors Gleevec (imatinib, Glivec) and Sutent (Sutinib, SU11248). Activating mutations also induce mastocytosis. Autocrine/paracrine stimulation may drive some lung and other tumors. Loss of expression associated with melanoma progression. Familial loss of function mutations cause piebaldism, with defects in hair and skin pigmentation due to lack of melanocytes.
UniProt Protein Details:	Protein type:EC 2.7.10.1; Protein kinase, TK; Protein kinase, tyrosine (receptor); Kinase, protein; Oncoprotein; Membrane protein, integral; TK group; PDGFR family Chromosomal Location of Human Ortholog: 4q12 Cellular Component: extracellular space; internal side of plasma membrane; mast cell granule; integral to membrane; acrosome; plasma membrane; intercellular junction; external side of plasma membrane Molecular Function:protein binding; protein homodimerization activity; protease binding; cytokine binding; metal ion binding; protein-tyrosine kinase activity; stem cell factor receptor activity; transmembrane receptor protein tyrosine kinase activity; receptor signaling protein tyrosine kinase activity; ATP binding Biological Process: nerve growth factor receptor signaling pathway; activation of MAPK activity; somatic stem cell maintenance; germ cell programmed cell death; positive regulation of JAK-STAT cascade; lymphoid progenitor cell differentiation; positive regulation of long-term neuronal synaptic plasticity; positive regulation of tyrosine phosphorylation of Stat3 protein; regulation of cell shape; epithelial cell proliferation; germ cell migration; somatic stem cell division; erythrocyte differentiation; T cell differentiation; fibroblast growth factor receptor signaling pathway; mast cell chemotaxis; embryonic hemopoiesis; stem cell differentiation; detection of mechanical stimulus involved in sensory perception of sound; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of tyrosine phosphorylation of Stat1 protein; immature B cell differentiation; regulation of pigmentation during development; glycosphingolipid metabolic process; positive regulation of transcription factor activity; mast cell cytokine production; peptidyl-tyrosine phosphorylation; protein amino acid autophosphorylation; signal transduction; ovarian follicle development; positive regulation of MAPKKK cascade; myeloid progenitor cell differentiation; melanocyte differentiation; positive regulation of cell proliferation; negative regulation of programmed cell death; visual learning; hemopoiesis; inflammatory response; positive regulation of Notch signaling pathway; lamellipodium biogenesis; epidermal growth factor receptor signaling pathway; phosphoinositide-mediated signaling; dendritic cell cytokine production; cytokine and chemokine mediated signaling pathway; male gonad development; stem cell maintenance; positive regulation of phosphoinositide 3-kinase activity; mast cell degranulation; regulation of cell proliferation; positive regulation of pseudopodium formation; positive regulation of tyrosine phosphorylation of Stat5 protein; gut development; pigmentation; actin cytoskeleton reorganization; innate immune response; spermatogenesis; spermatid development; positive regulation of cell migration Disease: Gastrointestinal Stromal Tumor; Mast Cell Disease; Piebald Trait; Testicular Germ Cell Tumor
NCBI Summary:	This gene encodes the human homolog of the proto-oncogene c-kit. C-kit was first identified as the cellular homolog of the feline sarcoma viral oncogene v-kit. This protein is a type 3 transmembrane receptor for MGF (mast cell growth factor, also known as stem cell factor). Mutations in this gene are associated with gastrointestinal stromal tumors, mast cell disease, acute myelogenous lukemia, and piebaldism. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P10721
NCBI GenInfo Identifier:	125472
NCBI Gene ID:	3815
NCBI Accession:	P10721.1
UniProt Secondary Accession:	P10721,Q6IQ28, Q99662, Q9UM99, B5A956, D5LXN2, D5M931 F5H8F8,
UniProt Related Accession:	P10721
Molecular Weight:
NCBI Full Name:	Mast/stem cell growth factor receptor Kit
NCBI Synonym Full Names:	v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
NCBI Official Symbol:	KIT
NCBI Official Synonym Symbols:	PBT; SCFR; C-Kit; CD117
NCBI Protein Information:	mast/stem cell growth factor receptor Kit; p145 c-kit; proto-oncogene c-Kit; piebald trait protein; soluble KIT variant 1; tyrosine-protein kinase Kit; proto-oncogene tyrosine-protein kinase Kit; v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene-like protein
UniProt Protein Name:	Mast/stem cell growth factor receptor Kit
UniProt Synonym Protein Names:	Piebald trait protein; PBT; Proto-oncogene c-Kit; Tyrosine-protein kinase Kit; p145 c-kit; v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; CD_antigen: CD117
Protein Family:	Mast/stem cell growth factor receptor
UniProt Gene Name:	KIT
UniProt Entry Name:	KIT_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-Kit (Phospho-Tyr721) Antibody, Anti-Kit 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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Kit (Phospho-Tyr721) Colorimetric Cell-Based ELISA Kit

Description