Ret (Phospho-Tyr1062)Colorimetric Cell-Based ELISA Kit (CBCAB00106)

Product Name:	Ret (Phospho-Tyr1062)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00106
ELISA Type:	Cell-Based
Target:	Ret (Phospho-Tyr1062)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Ret (Phospho-Tyr1062) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Ret protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Ret in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Ret phosphorylation.

Qualitative determination of Ret (Phospho-Tyr1062) concentration is achieved by an indirect ELISA format. In essence, Ret (Phospho-Tyr1062) is captured by Ret (Phospho-Tyr1062)-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: 5979, UniProt ID: P07949, OMIM: 114500/142623/155240/162300/164761/171300/171400/188550/191830/209880, Unigene: Hs.350321
Gene Symbol:	RET
Sub Type:	Phospho

UniProt Protein Function:	Ret: a proto-oncogenic receptor tyrosine kinase. Receptor for glial cell line-derived neurotropic factor (GDNF) and its congeners neurturin, persephin and artemin. Part of a multicompetent receptor complex with other membrane-bound ligand-binding GDNF family receptors (aGFRs). Required for development of the kidney and neural crest-derived cell types. Three alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:Protein kinase, TK; EC 2.7.10.1; Oncoprotein; Kinase, protein; Protein kinase, tyrosine (receptor); Membrane protein, integral; TK group; Ret family Chromosomal Location of Human Ortholog: 10q11.2 Cellular Component: cytoplasm; endosome membrane; integral to plasma membrane; intracellular membrane-bound organelle; plasma membrane; receptor complex Molecular Function:calcium ion binding; protein binding; protein-tyrosine kinase activity; Ras guanyl-nucleotide exchange factor activity; receptor activity Biological Process: MAPKKK cascade; membrane protein proteolysis; neuron adhesion; positive regulation of cell adhesion mediated by integrin; positive regulation of cell migration; positive regulation of transcription, DNA-dependent; posterior midgut development; protein amino acid phosphorylation; regulation of cell adhesion; response to pain; signal transduction Disease: Hirschsprung Disease, Susceptibility To, 1; Multiple Endocrine Neoplasia, Type Iia; Multiple Endocrine Neoplasia, Type Iib; Thyroid Carcinoma, Familial Medullary; Thyroid Carcinoma, Papillary
NCBI Summary:	This gene, a member of the cadherin superfamily, encodes one of the receptor tyrosine kinases, which are cell-surface molecules that transduce signals for cell growth and differentiation. This gene plays a crucial role in neural crest development, and it can undergo oncogenic activation in vivo and in vitro by cytogenetic rearrangement. Mutations in this gene are associated with the disorders multiple endocrine neoplasia, type IIA, multiple endocrine neoplasia, type IIB, Hirschsprung disease, and medullary thyroid carcinoma. Two transcript variants encoding different isoforms have been found for this gene. Additional transcript variants have been described but their biological validity has not been confirmed. [provided by RefSeq, Jul 2008]
UniProt Code:	P07949
NCBI GenInfo Identifier:	547807
NCBI Gene ID:	5979
NCBI Accession:	P07949.3
UniProt Secondary Accession:	P07949,Q15250, Q9BTB0, Q9H4A2, A8K6Z2,
UniProt Related Accession:	P07949
Molecular Weight:	119,847 Da
NCBI Full Name:	Proto-oncogene tyrosine-protein kinase receptor Ret
NCBI Synonym Full Names:	ret proto-oncogene
NCBI Official Symbol:	RET
NCBI Official Synonym Symbols:	PTC; MTC1; HSCR1; MEN2A; MEN2B; RET51; CDHF12; CDHR16; RET-ELE1
NCBI Protein Information:	proto-oncogene tyrosine-protein kinase receptor Ret
UniProt Protein Name:	Proto-oncogene tyrosine-protein kinase receptor Ret
UniProt Synonym Protein Names:	Cadherin family member 12; Proto-oncogene c-Ret
Protein Family:	Retbindin
UniProt Gene Name:	RET
UniProt Entry Name:	RET_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-Ret (Phospho-Tyr1062) Antibody, Anti-Ret 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.)