Figure 1: MMP2 Target Protein Structure.

MMP2 Introduction

Protein Function

• MMP2 is a matrix metalloproteinase capable of degrading the extracellular matrix, participating in various physiological processes such as vascular remodeling, angiogenesis, tissue repair, tumor invasion, inflammation, and rupture of atherosclerotic plaques.
• In addition to extracellular matrix proteins, MMP2 can act on non-matrix proteins such as endothelin-1 and calcitonin gene-related peptide (CGRP).
• The C-terminal non-catalytic fragment PEX of MMP2 exhibits anti-angiogenic and anti-tumor properties, inhibiting cell migration and reducing cell adhesion to basic fibroblast growth factor (FGF2) and fibronectin.
• Lack of MMP2 leads to reduced extracellular matrix degradation, thereby limiting macrophage migration to necrotic tissue and left ventricular rupture. Mutations in MMP2 are associated with Winchester syndrome and Nodulosis-Arthropathy-Osteolysis (NAO) syndrome.

Protein Expression

• MMP2 is found in the respiratory epithelial cells of most tissues, as well as in the placenta and in endothelial and stromal cells.
• PEX is expressed in various tumor tissues, such as gliomas, breast cancer, and prostate cancer.
• Aspirin may inhibit the expression of MMP2.

Protein Localization

• Human Isoform 1: Secreted into the extracellular matrix, cell membrane, and nucleus.
• Human Isoform 2: Cytoplasm; mitochondria.

Figure 2: ICC Experimental Results Image of MMP2 Protein, Anti-MMP2 Antibody [EPR1184] (ab92536). Green: MMP2; Red: alpha Tubulin; Blue: DAPI

Isoforms & Post-Translational Modifications

• Human (P08253):
  Isoform 1 (P08253-1): 73.8 kDa (predicted)
  Isoform 2 (P08253-2): 65.7 kDa (predicted)
  Isoform 3 (P08253-3): 68.8 kDa (predicted)
• Mouse (P33434):
  Isoform 1 (P33434-1): 74.1 kDa (predicted)
  Isoform 2 (P33434-2): 65.8 kDa (predicted)
• Rat (P33436): 74.1 kDa (predicted)
• Phosphorylation
• Glycosylation

WB Experiment Tips

Precautions

• Since MMP2 is mainly expressed in extracellular matrix fibroblasts, the expression level of the target protein in the sample must be confirmed before detection. We also advise conducting experiments with a positive control, as recommended in the antibody product manual.
• MMP2 is synthesized as a precursor form pro-MMP2 (72 kDa) and is secreted into the extracellular space. It is activated to form active MMP2 (62-69 kDa) through enzymatic cleavage. Therefore, multiple bands may appear in WB experiments.
• We recommend not cutting the membrane. For the experiment, the entire membrane, or at least the 50-100 kDa region, should be retained.

Positive Control

• Mouse NIH/3T3 cell lysate
• Human HT-1080 cell lysate

Negative Control (No or Weak Expression)

• Mouse liver tissue
• Rat spleen tissue

Example Results

Figure 3: WB Experimental Results of MMP2 Protein, Anti-MMP2 Antibody [EPR17003-25] (ab181286)

Lane 1: Mouse plasma lysate (20 µg)
Lane 2: Rat serum lysate (20 µg)
Lane 3: Mouse lung tissue lysate (20 µg)
Lane 4: Rat plasma lysate (20 µg)
Lane 5: Rat lung tissue lysate (20 µg)
Lane 6: HT1080 (human fibrosarcoma cells) whole cell lysate (10 µg)
Lane 7：Human plasma lysate (10 µg)

Predicted band size: 73 kDa
Detected band size: 72 kDa

Figure 4: WB Experimental Results of MMP2 Protein, Anti-MMP2 Antibody [EPR1184] (ab92536)

Lane 1: L6 (rat skeletal muscle cells) whole cell lysate, 1% SDS heat denaturation method (20 µg)
Lane 2: Mouse liver tissue lysate, RIPA lysis method (20 µg)
Lane 3: Mouse liver tissue lysate, 1% SDS heat denaturation method (20 µg)
Lane 4: Raw264.7 (mouse macrophage leukemia cells) whole cell lysate, RIPA lysis method (20 µg)
Lane 5: Raw264.7 (mouse macrophage leukemia cells) whole cell lysate, 1% SDS heat denaturation method (20 µg)
Lane 6: HepG2 (human liver cancer cells) whole cell lysate, RIPA lysis method (20 µg)
Lane 7: HepG2 (human liver cancer cells) whole cell lysate, 1% SDS heat denaturation method (20 µg)

Predicted band size: 74 kDa
Detected band sizes: 69 kDa, 72 kDa

Key Control Points

In addition to the routine issues that need to be paid attention to in the experiment, special attention should be paid to the following critical control points:

Sample preparation:

1. Add compound protease inhibitors to avoid degradation of target protein.
2. Select appropriate lysis buffer to enrich more target protein.
3. Ultrasonic disruption of cells to enrich target protein.
4. Keep samples on ice during the entire sample preparation process.
5. Determine the sample protein concentration by Bradford analysis, Lowry analysis or BCA analysis.

Blocking:

1. There is no blocking solution suitable for all systems, please choose a suitable blocking solution.

Antibody incubation:

1. During the WB experiment, please avoid dry membrane.
2. Please select the appropriate antibody working concentration according to the product manual.
3. It is recommended to use fresh antibodies and it is not recommended to reuse antibodies.

References

1. Busti C, Falcinelli E, Momi S, et al. Matrix metalloproteinases and peripheral arterial disease. Intern Emerg Med. (2010) 5:13–25. doi: https://doi.org/10.1007/s11739-009-0283-y
2. Ezhilarasan R, Jadhav U, Mohanan I et al. The hemopexin domain of MMP-9 inhibits angiogenesis and retards the growth of intracranial glioblastoma xenograft in nude mice. Int J Cancer. (2009) 124：306-315. doi: https://doi.org/10.1002/ijc.23951
3. Ganea E, Trifan M, Laslo AC, et al. Matrix metalloproteinases: useful and deleterious. Biochem Soc Trans. (2007) 35:989–991. doi: https://doi.org/10.1042/BST0350689
4. Zankl A, Bonafe L, Calcaterra V et al. Winchester syndrome caused by a homozygous mutation affecting the active site of matrix metalloproteinase 2. Clin Genet (2005) 67: 261–266. doi: https://doi.org/10.1111/j.1399-0004.2004.00402.x
5. Martignetti JA, Aqeel AA, Sewairi WA et al. Mutation of the matrix metalloproteinase 2 gene (MMP2) causes a multicentric osteolysis and arthritis syndrome. Nat Genet(2001) 28: 261–265. doi: https://doi.org/10.1038/90100