Description

Nori Canine MMP-1 ELISA Kit Summary

Alternative names for MMP-1: Matrix metalloproteinase-1 (MMP-1), interstitial collagenase, fibroblast collagenase

Alternative names for canine: Dog

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-1 (MMP-1) also known as interstitial collagenase and fibroblast collagenase is an enzyme that in humans is encoded by the MMP1 gene.^[1] MMP-1 has an archetypal structure consisting of a pre-domain, a pro-domain, a catalytic domain, a linker region and a hemopexin-like domain.^[2] The Catalytic Domains of MMPs share very similar characteristics, having a general shape of oblate ellipsoid with a diameter of ~40 Å. The Catalytic Domain of MMP-1 is composed of five highly twisted β-strands (sI-sV), three α-helix (hA-hC) and a total of eight loops, enclosing a total of five metal ions, three Ca²⁺ and two Zn²⁺, one of which with catalytic role. The Catalytic Domain (CAT) of MMP-1 starts with the F100 (non-truncated CAT) as the first amino-acid of the N-terminal loop of the CAT domain. A specific region (183)RWTNNFREY(191) has been identified as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity.^[3] On C-terminal part of the CAT Domain the hB α-helix, known as the “active-site helix” encompasses part of the “zinc-binding consensus sequence” HEXXHXXGXXH that is characteristic of the Metzincin superfamily.^[4] The α-helix hB finishes abruptly at Gly225 where the last loop of the domain starts. This last loop contains the “specificity loop” which is the shortest in the MMPs family. The Catalytic Domain ends at Gly261 with α-helix hC. MMPs 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. Specifically, MMP-1 breaks down the interstitial collagens, types I, II, and III. Mechanical force may increase the expression of MMP1 in human periodontal ligament cells.^[13] MMP1 has been shown to interact with CD49b.^[14][15]

References

1. Brinckerhoff CE, et al. (1987). Clin. Invest. 79 (2): 542–6.
2. Li J, et al. (1995). Structure 3 (6): 541–9.
3. Chung L, et al. (2000). Biol. Chem. 275 (38): 29610–7.
4. Bode W, et al. (1993). FEBS Lett. 331 (1-2): 134–40.
5. Huang SF, et al. (2008). J. Oral Sci. 116 (4): 319–23.mon
6. Dumin JA, et al. (2001). Biol. Chem. 276 (31): 29368–74.

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