EPHB1/2 (Phospho-Tyr594/604) Colorimetric Cell-Based ELISA Kit

Product Name:	EPHB1/2 (Phospho-Tyr594/604) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01420
ELISA Type:	Cell-Based
Target:	EPHB1/2 (Phospho-Tyr594/604)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The EPHB1/2 (Phospho-Tyr594/604) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect EPHB1/2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated EPHB1/2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on EPHB1/2 phosphorylation.

Qualitative determination of EPHB1/2 (Phospho-Tyr594/604) concentration is achieved by an indirect ELISA format. In essence, EPHB1/2 (Phospho-Tyr594/604) is captured by EPHB1/2 (Phospho-Tyr594/604)-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: 2047/1969, UniProt ID: P54762/P29317, OMIM: 600600/176946, Unigene: Hs.116092/Hs.171596
Gene Symbol:	EPHB1/EPHB2
Sub Type:	Phospho

UniProt Protein Function:	EphB1: a receptor tyrosine kinase of the Eph family. Receptor for members of the ephrin-B family: ephrin-B1, -B2 and -B3. The Eph receptor tyrosine kinase family, the largest in the tyrosine kinase group, has fourteen members. They bind membrane-anchored ligands, ephrins, at sites of cell-cell contact, regulating the repulsion and adhesion of cells that underlie the establishment, maintenance, and remodeling of patterns of cellular organization. Eph signals are particularly important in regulating cell adhesion and cell migration during development, axon guidance, homeostasis and disease. EphA receptors bind to GPI-anchored ephrin-A ligands, while EphB receptors bind to ephrin-B proteins that have a transmembrane and cytoplasmic domain. Interactions between EphB receptor kinases and ephrin-B proteins transduce signals bidirectionally, signaling to both interacting cell types. Eph receptors and ephrins also regulate the adhesion of endothelial cells and are required for the remodeling of blood vessels. The ligand-activated form of EphB1 interacts with GRB2, GRB10 and NCK through their respective SH2 domains. Four alternatively spliced isoforms are known.
UniProt Protein Details:	Protein type:Protein kinase, TK; Kinase, protein; EC 2.7.10.1; Protein kinase, tyrosine (receptor); Membrane protein, integral; TK group; Eph family Chromosomal Location of Human Ortholog: 3q21-q23 Cellular Component: cytosol; early endosome membrane; extracellular region; integral to plasma membrane; plasma membrane Molecular Function:protein binding; transmembrane-ephrin receptor activity Biological Process: angiogenesis; axon guidance; cell-substrate adhesion; central nervous system projection neuron axonogenesis; detection of temperature stimulus involved in sensory perception of pain; ephrin receptor signaling pathway; establishment of cell polarity; neurogenesis; positive regulation of synaptogenesis; protein amino acid autophosphorylation; regulation of JNK cascade; retinal ganglion cell axon guidance
NCBI Summary:	Ephrin receptors and their ligands, the ephrins, mediate numerous developmental processes, particularly in the nervous system. Based on their structures and sequence relationships, ephrins are divided into the ephrin-A (EFNA) class, which are anchored to the membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B (EFNB) class, which are transmembrane proteins. The Eph family of receptors are divided into 2 groups based on the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. Ephrin receptors make up the largest subgroup of the receptor tyrosine kinase (RTK) family. The protein encoded by this gene is a receptor for ephrin-B family members. [provided by RefSeq, Jul 2008]
UniProt Code:	P54762
NCBI GenInfo Identifier:	1706663
NCBI Gene ID:	2047
NCBI Accession:	P54762.1
UniProt Secondary Accession:	P54762,O43569, O95142, O95143, Q0VG87, A8K593, B3KTB2 B5A969,
UniProt Related Accession:	P54762
Molecular Weight:	26,906 Da
NCBI Full Name:	Ephrin type-B receptor 1
NCBI Synonym Full Names:	EPH receptor B1
NCBI Official Symbol:	EPHB1
NCBI Official Synonym Symbols:	ELK; NET; Hek6; EPHT2
NCBI Protein Information:	ephrin type-B receptor 1
UniProt Protein Name:	Ephrin type-B receptor 1
UniProt Synonym Protein Names:	ELK; EPH tyrosine kinase 2; EPH-like kinase 6; EK6; hEK6; Neuronally-expressed EPH-related tyrosine kinase; NET; Tyrosine-protein kinase receptor EPH-2
Protein Family:	Ephrin type-B receptor
UniProt Gene Name:	EPHB1
UniProt Entry Name:	EPHB1_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-EPHB1/2 (Phospho-Tyr594/604) Antibody, Anti-EPHB1/2 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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EPHB1/2 (Phospho-Tyr594/604) Colorimetric Cell-Based ELISA Kit

Description