TIMP2 Colorimetric Cell-Based ELISA Kit (CBCAB00885)

Product Name:	TIMP2 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00885
ELISA Type:	Cell-Based
Target:	TIMP2
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The TIMP2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TIMP2 protein expression profile in cells. The kit can be used for measuring the relative amounts of TIMP2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TIMP2.

Qualitative determination of TIMP2 concentration is achieved by an indirect ELISA format. In essence, TIMP2 is captured by TIMP2-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: 7077, UniProt ID: P16035, OMIM: 188825, Unigene: Hs.633514
Gene Symbol:	TIMP2
Sub Type:	None

UniProt Protein Function:	TIMP2: Complexes with metalloproteinases (such as collagenases) and irreversibly inactivates them by binding to their catalytic zinc cofactor. Known to act on MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, MMP-15, MMP-16 and MMP-19. Interacts (via the C-terminal) with MMP2 (via the C- terminal PEX domain); the interaction inhibits the MMP2 activity. Down-regulated by TGFB1. Belongs to the protease inhibitor I35 (TIMP) family.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide; Extracellular matrix; Motility/polarity/chemotaxis; Cell development/differentiation; Inhibitor Chromosomal Location of Human Ortholog: 17q25 Cellular Component: extracellular space; proteinaceous extracellular matrix; growth cone; cell surface; cell soma; extracellular region; basement membrane Molecular Function:integrin binding; metalloendopeptidase inhibitor activity; protein binding; protease binding; metal ion binding; enzyme activator activity Biological Process: response to drug; regulation of Rap protein signal transduction; negative regulation of metalloenzyme activity; extracellular matrix organization and biogenesis; central nervous system development; response to hormone stimulus; negative regulation of membrane protein ectodomain proteolysis; extracellular matrix disassembly; negative regulation of cell proliferation; positive regulation of MAPKKK cascade; response to cytokine stimulus; negative regulation of Ras protein signal transduction; negative regulation of mitotic cell cycle; positive regulation of adenylate cyclase activity; positive regulation of neuron differentiation; aging
NCBI Summary:	This gene is a member of the TIMP gene family. The proteins encoded by this gene family are natural inhibitors of the matrix metalloproteinases, a group of peptidases involved in degradation of the extracellular matrix. In addition to an inhibitory role against metalloproteinases, the encoded protein has a unique role among TIMP family members in its ability to directly suppress the proliferation of endothelial cells. As a result, the encoded protein may be critical to the maintenance of tissue homeostasis by suppressing the proliferation of quiescent tissues in response to angiogenic factors, and by inhibiting protease activity in tissues undergoing remodelling of the extracellular matrix. [provided by RefSeq, Jul 2008]
UniProt Code:	P16035
NCBI GenInfo Identifier:	135854
NCBI Gene ID:	7077
NCBI Accession:	P16035.2
UniProt Secondary Accession:	P16035,Q16121, Q93006, Q9UDF7,
UniProt Related Accession:	P16035,Q96MC4
Molecular Weight:	24,399 Da
NCBI Full Name:	Metalloproteinase inhibitor 2
NCBI Synonym Full Names:	TIMP metallopeptidase inhibitor 2
NCBI Official Symbol:	TIMP2
NCBI Official Synonym Symbols:	DDC8; CSC-21K
NCBI Protein Information:	metalloproteinase inhibitor 2; TIMP-2; tissue inhibitor of metalloproteinase 2; tissue inhibitor of metalloproteinases 2
UniProt Protein Name:	Metalloproteinase inhibitor 2
UniProt Synonym Protein Names:	CSC-21K; Tissue inhibitor of metalloproteinases 2; TIMP-2
Protein Family:	Metalloproteinase inhibitor
UniProt Gene Name:	TIMP2
UniProt Entry Name:	TIMP2_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-TIMP2 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.)