Integrin beta1 (Phospho-Thr789) Colorimetric Cell-Based ELISA Kit

Product Name:	Integrin beta1 (Phospho-Thr789) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01293
ELISA Type:	Cell-Based
Target:	Integrin beta1 (Phospho-Thr789)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Integrin beta1 (Phospho-Thr789) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Integrin beta1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Integrin beta1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Integrin beta1 phosphorylation.

Qualitative determination of Integrin beta1 (Phospho-Thr789) concentration is achieved by an indirect ELISA format. In essence, Integrin beta1 (Phospho-Thr789) is captured by Integrin beta1 (Phospho-Thr789)-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: 3688, UniProt ID: P05556, OMIM: 135630, Unigene: Hs.713531
Gene Symbol:	ITGB1
Sub Type:	Phospho

UniProt Protein Function:	ITGB1: an integral membrane protein that heterodimerizes with an alpha-3 chain, forming a receptor for many extracellular-matrix proteins including fibronectin, laminin, collagen, epiligrin and thrombospondin.. Beta 1 integrins recognize the amino-acid motif RGD in a wide array of ligands. Five alternatively spliced variants with alternate carboxy termini have been described. Two alternatively spliced isoforms have been described. Isoform beta-1a is widely expressed; other isoforms are generally expressed with a more restricted distribution. Isoform beta-1b is expressed in skin, liver, skeletal muscle, cardiac muscle, placenta, umbilical vein endothelial cells, neuroblastoma cells, lymphoma cells, hepatoma cells and astrocytoma cells. Isoforms beta-1c and beta-1c-2 are expressed in muscle, kidney, liver, placenta, cervical epithelium, umbilical vein endothelial cells, fibroblast cells, embryonal kidney cells, platelets and several blood cell lines. Isoform beta-c-2, rather than isoform beta-1c, is selectively expressed in primary t-cells. Isoform beta-1c is expressed in nonproliferating and differentiated prostate gland epithelial cells. Isoform beta-1d is expressed specifically in striated muscle (skeletal and cardiac muscle).
UniProt Protein Details:	Protein type:Cell adhesion; Cell surface; Membrane protein, integral; Motility/polarity/chemotaxis; Receptor, misc. Chromosomal Location of Human Ortholog: 10p11.22 Cellular Component: cell surface; cell-cell adherens junction; cytoplasm; filopodium; focal adhesion; lipid raft; membrane; neuromuscular junction; plasma membrane; receptor complex; ruffle; sarcolemma Molecular Function:actin binding; C-X3-C chemokine binding; cell adhesion molecule binding; coreceptor activity; fibronectin binding; protease binding; protein binding; protein complex binding; protein heterodimerization activity Biological Process: B cell differentiation; calcium-independent cell-matrix adhesion; cell adhesion mediated by integrin; cell migration; cell-cell adhesion mediated by integrin; cell-matrix adhesion; cell-substrate adhesion; cellular defense response; extracellular matrix organization and biogenesis; heterotypic cell-cell adhesion; homophilic cell adhesion; integrin-mediated signaling pathway; leukocyte adhesion; leukocyte migration; leukocyte tethering or rolling; mesodermal cell differentiation; positive regulation of apoptosis; positive regulation of GTPase activity; receptor internalization; regulation of immune response; stress fiber formation; transforming growth factor beta receptor signaling pathway
NCBI Summary:	Integrins are heterodimeric proteins made up of alpha and beta subunits. At least 18 alpha and 8 beta subunits have been described in mammals. Integrin family members are membrane receptors involved in cell adhesion and recognition in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic diffusion of tumor cells. This gene encodes a beta subunit. Multiple alternatively spliced transcript variants which encode different protein isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P05556
NCBI GenInfo Identifier:	218563324
NCBI Gene ID:	3688
NCBI Accession:	P05556.2
UniProt Secondary Accession:	P05556,P78466, P78467, Q13089, Q13090, Q13091, Q13212 A8K6N2, D3DRX9, D3DRY3, D3DRY4, D3DRY5,
UniProt Related Accession:	P05556
Molecular Weight:	88,884 Da
NCBI Full Name:	Integrin beta-1
NCBI Synonym Full Names:	integrin subunit beta 1
NCBI Official Symbol:	ITGB1
NCBI Official Synonym Symbols:	CD29; FNRB; MDF2; VLAB; GPIIA; MSK12; VLA-BETA
NCBI Protein Information:	integrin beta-1
UniProt Protein Name:	Integrin beta-1
UniProt Synonym Protein Names:	Fibronectin receptor subunit beta; Glycoprotein IIa; GPIIA; VLA-4 subunit beta; CD_antigen: CD29
UniProt Gene Name:	ITGB1

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 beta1 (Phospho-Thr789) Antibody, Anti-Integrin beta1 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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Integrin beta1 (Phospho-Thr789) Colorimetric Cell-Based ELISA Kit

Description