BCAR1 Colorimetric Cell-Based ELISA Kit (CBCAB00542)

Product Name:	BCAR1 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00542
ELISA Type:	Cell-Based
Target:	BCAR1
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The BCAR1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect BCAR1 protein expression profile in cells. The kit can be used for measuring the relative amounts of BCAR1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on BCAR1.

Qualitative determination of BCAR1 concentration is achieved by an indirect ELISA format. In essence, BCAR1 is captured by BCAR1-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: 9564, UniProt ID: P56945, OMIM: 602941, Unigene: Hs.479747
Gene Symbol:	BCAR1
Sub Type:	None

UniProt Protein Function:	P130Cas: a docking protein containing multiple protein-protein interaction domains. Plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. Implicated in induction of cell migration. The amino-terminal SH3 domain regulates its interaction with focal adhesion kinase (FAK) and the FAK-related kinase PYK2 and also with tyrosine phosphatases PTP-1B and PTP-PEST. Overexpression confers antiestrogen resistance on breast cancer cells.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Adaptor/scaffold Chromosomal Location of Human Ortholog: 16q23.1 Cellular Component: ruffle; focal adhesion; lamellipodium; cytoplasm; nucleolus; plasma membrane; cytosol; actin cytoskeleton Molecular Function:signal transducer activity; protein binding; SH3 domain binding; protein kinase binding Biological Process: epidermal growth factor receptor signaling pathway; integrin-mediated signaling pathway; antigen receptor-mediated signaling pathway; platelet activation; cell migration; nerve growth factor receptor signaling pathway; actin filament organization; hepatocyte growth factor receptor signaling pathway; platelet-derived growth factor receptor signaling pathway; T cell receptor signaling pathway; regulation of apoptosis; G-protein coupled receptor protein signaling pathway; cell proliferation; B cell receptor signaling pathway; cell division; insulin receptor signaling pathway; regulation of cell growth; cell adhesion; vascular endothelial growth factor receptor signaling pathway; blood coagulation; positive regulation of cell migration
NCBI Summary:	BCAR1, or CAS, is an Src (MIM 190090) family kinase substrate involved in various cellular events, including migration, survival, transformation, and invasion (Sawada et al., 2006 [PubMed 17129785]).[supplied by OMIM, May 2009]
UniProt Code:	P56945
NCBI GenInfo Identifier:	288558806
NCBI Gene ID:	9564
NCBI Accession:	P56945.2
UniProt Secondary Accession:	P56945,B3KWD7, B4DEV4, B4DGB5, B4DIW5, B7Z7X7, E9PCL5 E9PCV2, F5GXA2, F5GXV6, F5H7Z0, F8WA69,
UniProt Related Accession:	P56945
Molecular Weight:	870
NCBI Full Name:	Breast cancer anti-estrogen resistance protein 1
NCBI Synonym Full Names:	breast cancer anti-estrogen resistance 1
NCBI Official Symbol:	BCAR1
NCBI Official Synonym Symbols:	CAS; CAS1; CASS1; CRKAS; P130Cas
NCBI Protein Information:	breast cancer anti-estrogen resistance protein 1; Crk-associated substrate p130Cas; Cas scaffolding protein family member 1
UniProt Protein Name:	Breast cancer anti-estrogen resistance protein 1
UniProt Synonym Protein Names:	CRK-associated substrate; Cas scaffolding protein family member 1; p130cas
Protein Family:	Breast cancer anti-estrogen resistance protein
UniProt Gene Name:	BCAR1
UniProt Entry Name:	BCAR1_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-BCAR1 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.)