Integrin beta4 (Phospho-Tyr1510) Cell-Based ELISA Kit

Product Name:	Integrin beta4 (Phospho-Tyr1510)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01458
ELISA Type:	Cell-Based
Target:	Integrin beta4 (Phospho-Tyr1510)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Integrin beta4 (Phospho-Tyr1510) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Integrin beta4 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Integrin beta4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Integrin beta4 phosphorylation.

Qualitative determination of Integrin beta4 (Phospho-Tyr1510) concentration is achieved by an indirect ELISA format. In essence, Integrin beta4 (Phospho-Tyr1510) is captured by Integrin beta4 (Phospho-Tyr1510)-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: 3691, UniProt ID: P16144, OMIM: 147557/226650/226730, Unigene: Hs.407966/Hs.632226
Gene Symbol:	ITGB4
Sub Type:	Phospho

UniProt Protein Function:	ITGB4: integrin alpha-6/beta-4 is a receptor for laminin. It plays a critical structural role in the hemidesmosome of epithelial cells. Five alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Cell adhesion; Membrane protein, integral; Receptor, misc. Chromosomal Location of Human Ortholog: 17q25 Cellular Component: cell surface; hemidesmosome; leading edge; plasma membrane; basal plasma membrane; basement membrane; integrin complex; receptor complex Molecular Function:protein binding; G-protein-coupled receptor binding; receptor activity Biological Process: skin development; integrin-mediated signaling pathway; extracellular matrix organization and biogenesis; hemidesmosome assembly; filopodium formation; gut development; cell-matrix adhesion; cell motility involved in cell locomotion; response to wounding; mesodermal cell differentiation; autophagy; cell adhesion Disease: Epidermolysis Bullosa, Junctional, Non-herlitz Type; Epidermolysis Bullosa Simplex, Localized; Epidermolysis Bullosa Junctionalis With Pyloric Atresia
NCBI Summary:	Integrins are heterodimers comprised of alpha and beta subunits, that are noncovalently associated transmembrane glycoprotein receptors. Different combinations of alpha and beta polypeptides form complexes that vary in their ligand-binding specificities. Integrins mediate cell-matrix or cell-cell adhesion, and transduced signals that regulate gene expression and cell growth. This gene encodes the integrin beta 4 subunit, a receptor for the laminins. This subunit tends to associate with alpha 6 subunit and is likely to play a pivotal role in the biology of invasive carcinoma. Mutations in this gene are associated with epidermolysis bullosa with pyloric atresia. Multiple alternatively spliced transcript variants encoding distinct isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P16144
NCBI GenInfo Identifier:	317373584
NCBI Gene ID:	3691
NCBI Accession:	P16144.5
UniProt Secondary Accession:	P16144,O14690, O14691, O15339, O15340, O15341, Q0VF97 Q9UIQ4, A0AVL6,
UniProt Related Accession:	P16144
Molecular Weight:	106,779 Da
NCBI Full Name:	Integrin beta-4
NCBI Synonym Full Names:	integrin, beta 4
NCBI Official Symbol:	ITGB4
NCBI Official Synonym Symbols:	CD104
NCBI Protein Information:	integrin beta-4; GP150; CD104 antigen; integrin beta-4 subunit
UniProt Protein Name:	Integrin beta-4
UniProt Synonym Protein Names:	GP150; CD_antigen: CD104
Protein Family:	Integrin
UniProt Gene Name:	ITGB4
UniProt Entry Name:	ITB4_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-Integrin beta4 (Phospho-Tyr1510) Antibody, Anti-Integrin beta4 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.)