Description

Nori Feline MMP-2 ELISA Kit Summary

Alternative names for MMP-2: Matrix metalloproteinase-2 (MMP-2), 72 kDa type IV collagenase, gelatinase A

Alternative name for feline: cat

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Matrix metalloproteinase-2 (MMP-2) is known as 72 kDa type IV collagenase, and gelatinase A is an enzyme that in humans is encoded by the MMP2 gene.(1) Proteins of the 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. This gene encodes an enzyme which degrades type IV collagen, the major structural component of basement membranes. The enzyme plays a role in endometrial menstrual breakdown, regulation of vascularization and the inflammatory response. Activation of MMP-2 requires a proteolytic processing. A complex of membrane type 1 MMP (MT1-MMP/MMP14) and tissue inhibitor of metalloproteinase 2 recruits pro-MMP 2 from extracellular milie to the cell surface. Activation then requires active molecule of MT1-MMP and auto catalytic cleavage. Clustering of integrin chain promotes activation of MMP-2. Another factor that will support the activation of MMP-2 is cell-cell clustering. A wild-type activated leukocyte cell adhesion molecule (ALCAM) also required to activate MMP-2. Mutations in the MMP2 gene are associated with Torg-Winchester syndrome, multicentric osteolysis and arthritis syndrome.(2) MMP2 has been shown to interact with THBS2,(3) TIMP2,(4-7) Thrombospondin 1,(4) CCL7 (8) and TIMP4.(6, 7)

Reference

1. Devarajan P, et al. Biol. Chem. 267 (35): 25228–32.
2. Martignetti JA, et al. Genet. 28 (3): 261–5.
3. Bein K, Simons M. Biol. Chem. 275 (41): 32167–73.
4. Morgunova E, et al. Natl. Acad. Sci. U.S.A. 99 (11): 7414–9.
5. Overall CM, et al. Biol. Chem. 275 (50): 39497–506.
6. Bigg HF, et al. Biol. Chem. 272 (24): 15496–500.
7. Kai HS, et al. Biol. Chem. 277 (50): 48696–707.
8. McQuibban GA, et al. Science 289 (5482): 1202–6.

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