BCAR3 Colorimetric Cell-Based ELISA (CBCAB00954)

Product Name:	BCAR3 Colorimetric Cell-Based ELISA
Product Code:	CBCAB00954
ELISA Type:	Cell-Based
Target:	BCAR3
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The BCAR3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect BCAR3 protein expression profile in cells. The kit can be used for measuring the relative amounts of BCAR3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on BCAR3.

Qualitative determination of BCAR3 concentration is achieved by an indirect ELISA format. In essence, BCAR3 is captured by BCAR3-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: 8412, UniProt ID: O75815, OMIM: 604704, Unigene: Hs.36958
Gene Symbol:	BCAR3
Sub Type:	None

UniProt Protein Function:	BCAR3: a ubiquitously expressed regulator of R-Ras activity that has been implicated in breast cancer. A GDP exchange factor whose overexpression confers antiestrogen resistance, activates Rac, PAK1, and the cyclin D1. Can accelerate the degradation of IRS-1, thus regulating signaling through the insulin receptor. When overexpressed, it confers anti-estrogen resistance in breast cancer cell lines. May also be regulated by cellular adhesion to extracellular matrix proteins. Up-regulated by interleukin-1-alpha and TNF-beta in thymus cortical reticular cell lines (mouse).Two alternatively spliced human isoforms have been described.
UniProt Protein Details:	Protein type:GEFs; Adaptor/scaffold; Motility/polarity/chemotaxis; GEFs, Ras Chromosomal Location of Human Ortholog: 1p22.1 Cellular Component: intracellular Molecular Function:guanyl-nucleotide exchange factor activity; protein binding Biological Process: lens morphogenesis in camera-type eye; positive regulation of GTPase activity; positive regulation of peptidyl-serine phosphorylation; response to drug; signal transduction; small GTPase mediated signal transduction
NCBI Summary:	Breast tumors are initially dependent on estrogens for growth and progression and can be inhibited by anti-estrogens such as tamoxifen. However, breast cancers progress to become anti-estrogen resistant. Breast cancer anti-estrogen resistance gene 3 was identified in the search for genes involved in the development of estrogen resistance. The gene encodes a component of intracellular signal transduction that causes estrogen-independent proliferation in human breast cancer cells. The protein contains a putative src homology 2 (SH2) domain, a hall mark of cellular tyrosine kinase signaling molecules, and is partly homologous to the cell division cycle protein CDC48. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2012]
UniProt Code:	O75815
NCBI GenInfo Identifier:	74739634
NCBI Gene ID:	8412
NCBI Accession:	O75815.1
UniProt Secondary Accession:	O75815,Q5TEW3, Q6UW40, Q9BR50, D3DT43,
UniProt Related Accession:	O75815
Molecular Weight:	82,357 Da
NCBI Full Name:	Breast cancer anti-estrogen resistance protein 3
NCBI Synonym Full Names:	breast cancer anti-estrogen resistance 3
NCBI Official Symbol:	BCAR3
NCBI Official Synonym Symbols:	NSP2; SH2D3B
NCBI Protein Information:	breast cancer anti-estrogen resistance protein 3
UniProt Protein Name:	Breast cancer anti-estrogen resistance protein 3
UniProt Synonym Protein Names:	Novel SH2-containing protein 2; SH2 domain-containing protein 3B
Protein Family:	Breast cancer anti-estrogen resistance protein
UniProt Gene Name:	BCAR3
UniProt Entry Name:	BCAR3_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-BCAR3 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.)