CD147 Colorimetric Cell-Based ELISA (CBCAB01006)

Product Name:	CD147 Colorimetric Cell-Based ELISA
Product Code:	CBCAB01006
ELISA Type:	Cell-Based
Target:	CD147
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The CD147 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CD147 protein expression profile in cells. The kit can be used for measuring the relative amounts of CD147 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on CD147.

Qualitative determination of CD147 concentration is achieved by an indirect ELISA format. In essence, CD147 is captured by CD147-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: 682, UniProt ID: P35613, OMIM: 109480/111380, Unigene: Hs.501293
Gene Symbol:	BSG
Sub Type:	None

UniProt Protein Function:	BSG: Plays pivotal roles in spermatogenesis, embryo implantation, neural network formation and tumor progression. Stimulates adjacent fibroblasts to produce matrix metalloproteinases (MMPS). May target monocarboxylate transporters SLC16A1, SLC16A3 and SLC16A8 to plasma membranes of retinal pigment epithelium and neural retina. Seems to be a receptor for oligomannosidic glycans. In vitro, promotes outgrowth of astrocytic processes. Enriched on the surface of tumor cells. Up-regulated in gliomas. Its expression levels correlate with malignant potential of the tumor. Forms homooligomers in a cis-dependent manner on the plasma membrane. Forms a complex with MMP1 at the tumor cell surface. Interacts with SLC16A1 and SLC1A3; probably a BSG dimer is associated with a monocarboxylate transporter dimer. Interacts with ATP1B2, MAG and L1CAM. Interacts with AJAP1. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, integral; Immunoglobulin superfamily Chromosomal Location of Human Ortholog: 19p13.3 Cellular Component: acrosomal membrane; focal adhesion; Golgi membrane; integral to plasma membrane; lipid raft; melanosome; membrane; mitochondrion; plasma membrane; sarcolemma Molecular Function:mannose binding; protein binding Biological Process: blood coagulation; cell surface receptor linked signal transduction; cellular metabolic process; decidualization; embryo implantation; extracellular matrix disassembly; extracellular matrix organization and biogenesis; leukocyte migration; odontogenesis of dentine-containing teeth; pyruvate metabolic process; response to cAMP; response to mercury ion; response to peptide hormone stimulus Disease: Blood Group--ok
NCBI Summary:	The protein encoded by this gene is a plasma membrane protein that is important in spermatogenesis, embryo implantation, neural network formation, and tumor progression. The encoded protein is also a member of the immunoglobulin superfamily. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P35613
NCBI GenInfo Identifier:	51704273
NCBI Gene ID:	682
NCBI Accession:	P35613.2
UniProt Secondary Accession:	P35613,Q7Z796, Q8IZL7, A6NJW1, D3YLG5,
UniProt Related Accession:	P35613
Molecular Weight:	22,764 Da
NCBI Full Name:	Basigin
NCBI Synonym Full Names:	basigin (Ok blood group)
NCBI Official Symbol:	BSG
NCBI Official Synonym Symbols:	OK; 5F7; TCSF; CD147; EMMPRIN
NCBI Protein Information:	basigin
UniProt Protein Name:	Basigin
UniProt Synonym Protein Names:	5F7; Collagenase stimulatory factor; Extracellular matrix metalloproteinase inducer; EMMPRIN; Leukocyte activation antigen M6; OK blood group antigen; Tumor cell-derived collagenase stimulatory factor; TCSF; CD_antigen: CD147
Protein Family:	Basigin
UniProt Gene Name:	BSG
UniProt Entry Name:	BASI_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-CD147 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.)

CD147 Colorimetric Cell-Based ELISA (CBCAB01006)

Description