MMP-14 Colorimetric Cell-Based ELISA Kit (CBCAB00128)

Product Name:	MMP-14 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00128
ELISA Type:	Cell-Based
Target:	MMP-14
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The MMP-14 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MMP-14 protein expression profile in cells. The kit can be used for measuring the relative amounts of MMP-14 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MMP-14.

Qualitative determination of MMP-14 concentration is achieved by an indirect ELISA format. In essence, MMP-14 is captured by MMP-14-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: 4323, UniProt ID: P50281, OMIM: 600754, Unigene: Hs.2399
Gene Symbol:	MMP14
Sub Type:	None

UniProt Protein Function:	MMP14: Seems to specifically activate progelatinase A. May thus trigger invasion by tumor cells by activating progelatinase A on the tumor cell surface. May be involved in actin cytoskeleton reorganization by cleaving PTK7. Up-regulated by NANOS1. Expressed in stromal cells of colon, breast, and head and neck. Expressed in lung tumors. Belongs to the peptidase M10A family.
UniProt Protein Details:	Protein type:EC 3.4.24.80; Protease; Motility/polarity/chemotaxis; Membrane protein, integral Chromosomal Location of Human Ortholog: 14q11.2 Cellular Component: extracellular matrix; focal adhesion; Golgi lumen; integral to plasma membrane; cytoplasm; melanosome; plasma membrane Molecular Function:integrin binding; protein binding; protease activator activity; zinc ion binding; metalloendopeptidase activity; calcium ion binding; transcription factor activity Biological Process: extracellular matrix organization and biogenesis; tissue remodeling; negative regulation of focal adhesion formation; male gonad development; embryonic cranial skeleton morphogenesis; proteolysis; positive regulation of cell growth; collagen catabolic process; extracellular matrix disassembly; regulation of transcription, DNA-dependent; branching morphogenesis of a tube; ovarian follicle development; response to estrogen stimulus; response to mechanical stimulus; endothelial cell proliferation; response to hypoxia; zymogen activation; response to oxidative stress; angiogenesis; astrocyte cell migration; lung development; positive regulation of cell migration; endochondral ossification Disease: Winchester Syndrome
NCBI Summary:	Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. However, the protein encoded by this gene is a member of the membrane-type MMP (MT-MMP) subfamily; each member of this subfamily contains a potential transmembrane domain suggesting that these proteins are expressed at the cell surface rather than secreted. This protein activates MMP2 protein, and this activity may be involved in tumor invasion. [provided by RefSeq, Jul 2008]
UniProt Code:	P50281
NCBI GenInfo Identifier:	317373419
NCBI Gene ID:	4323
NCBI Accession:	P50281.3
UniProt Secondary Accession:	P50281,Q6GSF3, Q92678, A8K5L0,
UniProt Related Accession:	P50281
Molecular Weight:	65,894 Da
NCBI Full Name:	Matrix metalloproteinase-14
NCBI Synonym Full Names:	matrix metallopeptidase 14 (membrane-inserted)
NCBI Official Symbol:	MMP14
NCBI Official Synonym Symbols:	MMP-14; MMP-X1; MT-MMP; MT1MMP; MTMMP1; WNCHRS; MT1-MMP; MT-MMP 1
NCBI Protein Information:	matrix metalloproteinase-14; membrane type 1 metalloprotease; membrane-type-1 matrix metalloproteinase
UniProt Protein Name:	Matrix metalloproteinase-14
UniProt Synonym Protein Names:	MMP-X1; Membrane-type matrix metalloproteinase 1; MT-MMP 1; MTMMP1; Membrane-type-1 matrix metalloproteinase; MT1-MMP; MT1MMP
Protein Family:	Matrix metalloproteinase
UniProt Gene Name:	MMP14
UniProt Entry Name:	MMP14_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-MMP-14 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.)