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MMP-2 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00764
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cardiovascular
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

MMP-2 Colorimetric Cell-Based ELISA Kit

The MMP-2 Colorimetric Cell-Based ELISA Kit is a powerful tool for the detection and quantification of MMP-2 levels in cell culture samples. With its high sensitivity and specificity, this kit provides accurate and reproducible results, making it essential for researchers studying cell migration, tissue remodeling, and cancer metastasis.MMP-2, also known as gelatinase A, is a key enzyme that plays a crucial role in extracellular matrix degradation and remodeling. Abnormal MMP-2 activity has been linked to various diseases, including cancer, arthritis, and cardiovascular disorders.

By measuring MMP-2 levels in cell cultures, researchers can gain valuable insights into disease mechanisms and potential therapeutic targets.The MMP-2 Colorimetric Cell-Based ELISA Kit is easy to use and provides rapid results, making it a valuable asset for any laboratory conducting research on MMP-2 biology and its implications in human health. Order yours today and unlock the potential of MMP-2 research in your studies.

Product Name:MMP-2 Colorimetric Cell-Based ELISA
Product Code:CBCAB00764
ELISA Type:Cell-Based
Target:MMP-2
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The MMP-2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MMP-2 protein expression profile in cells. The kit can be used for measuring the relative amounts of MMP-2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MMP-2.

Qualitative determination of MMP-2 concentration is achieved by an indirect ELISA format. In essence, MMP-2 is captured by MMP-2-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: 4313, UniProt ID: P08253, OMIM: 120360/259600, Unigene: Hs.513617
Gene Symbol:MMP2
Sub Type:None
UniProt Protein Function:MMP2: Ubiquitinous metalloproteinase that is involved in diverse functions such as remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. As well as degrading extracellular matrix proteins, can also act on several nonmatrix proteins such as big endothelial 1 and beta-type CGRP promoting vasoconstriction. Also cleaves KISS at a Gly-|-Leu bond. Appears to have a role in myocardial cell death pathways. Contributes to myocardial oxidative stress by regulating the activity of GSK3beta. Cleaves GSK3beta in vitro. Interacts (via the C-terminal hemopexin-like domains- containing region) with the integrin alpha-V/beta-3; the interaction promotes vascular invasion in angiogenic vessels and melamoma cells. Interacts (via the C-terminal PEX domain) with TIMP2 (via the C-terminal); the interaction inhibits the degradation activity. Interacts with GSK3B. Aspirin appears to inhibit expression. Produced by normal skin fibroblasts. PEX is expressed in a number of tumors including gliomas, breast and prostate. Inhibited by histatin-3 1/24 (histatin-5). Belongs to the peptidase M10A family.
UniProt Protein Details:

Protein type:Secreted; Motility/polarity/chemotaxis; Cell development/differentiation; Protease; Apoptosis; EC 3.4.24.24; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 16q12.2

Cellular Component: extracellular space; proteinaceous extracellular matrix; sarcomere; mitochondrion; extracellular region; plasma membrane; nucleus

Molecular Function:protein binding; zinc ion binding; serine-type endopeptidase activity; metalloendopeptidase activity

Biological Process: extracellular matrix disassembly; collagen catabolic process; axon guidance; extracellular matrix organization and biogenesis; intramembranous ossification; cellular protein metabolic process; positive regulation of innate immune response; ephrin receptor signaling pathway; response to hypoxia; angiogenesis; proteolysis; blood vessel maturation; embryo implantation

Disease: Multicentric Osteolysis, Nodulosis, And Arthropathy

NCBI Summary:This gene is a member of the matrix metalloproteinase (MMP) gene family, that are zinc-dependent enzymes capable of cleaving components of the extracellular matrix and molecules involved in signal transduction. The protein encoded by this gene is a gelatinase A, type IV collagenase, that contains three fibronectin type II repeats in its catalytic site that allow binding of denatured type IV and V collagen and elastin. Unlike most MMP family members, activation of this protein can occur on the cell membrane. This enzyme can be activated extracellularly by proteases, or, intracellulary by its S-glutathiolation with no requirement for proteolytical removal of the pro-domain. This protein is thought to be involved in multiple pathways including roles in the nervous system, endometrial menstrual breakdown, regulation of vascularization, and metastasis. Mutations in this gene have been associated with Winchester syndrome and Nodulosis-Arthropathy-Osteolysis (NAO) syndrome. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Oct 2014]
UniProt Code:P08253
NCBI GenInfo Identifier:116856
NCBI Gene ID:4313
NCBI Accession:P08253.2
UniProt Secondary Accession:P08253,Q9UCJ8, B2R6U1, B4DWH3, E9PE45,
UniProt Related Accession:P08253
Molecular Weight:68,831 Da
NCBI Full Name:72 kDa type IV collagenase
NCBI Synonym Full Names:matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase)
NCBI Official Symbol:MMP2  
NCBI Official Synonym Symbols:CLG4; MONA; CLG4A; MMP-2; TBE-1; MMP-II  
NCBI Protein Information:72 kDa type IV collagenase; gelatinase A; 72 kDa gelatinase; collagenase type IV-A; neutrophil gelatinase; matrix metalloproteinase-2; matrix metalloproteinase-II
UniProt Protein Name:72 kDa type IV collagenase
UniProt Synonym Protein Names:72 kDa gelatinase; Gelatinase A; Matrix metalloproteinase-2; MMP-2; TBE-1Cleaved into the following chain:PEX
Protein Family:Matrix metalloproteinase
UniProt Gene Name:MMP2  
UniProt Entry Name:MMP2_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 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.

StepProcedure
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-2 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.)

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