Figure 1: Protein structure of MMP13 target.

MMP13 Introduction

Protein Function

• MMP13 is a member of the matrix metalloproteinase (MMP) peptidase M10 family, capable of degrading various extracellular matrix proteins, including fibrillar collagens, fibronectin, tenascin-C (TNC), and aggrecan (ACAN).
• MMP13 exhibits higher activity in cleaving triple-helical collagens, favoring type II collagen over type I and III collagens. Additionally, MMP13 degrades collagen types IV, XIV, and X.
• MMP13 plays roles in various physiological processes such as wound healing, tissue remodeling, cartilage degradation, skeletal development, bone mineralization, and ossification.
• MMP13 is implicated in cell migration and tumor cell invasion, with its gene mutations associated with metaphyseal dysplasia.

Protein Expression

• Detected in fetal cartilage and cranial bones.
• Detected in chondrocytes of hypertrophic cartilage in vertebrae, ossified distal ends of ribs, and osteoblasts and periosteal cells beneath ossified ribs.
• It was detected in cartilage cells of joint cartilage treated with TNF and IL-1β but not in untreated cartilage cells.
• Detectable in T lymphocytes.
• Detected in breast cancer tissues.
• Expression of MMP13 is upregulated by TNF and IL-1β induction.

Protein Localization

• Secreted into the extracellular matrix or extracellular space.

Isoforms & Post-translational modifications

• Human (P45452): 53.8 kDa (predicted)
• Mouse (P33435): 54.1 kDa (predicted)
• Rat (P23097): 53.3 kDa (predicted)
• Pro-MMP13 is activated by removing the propeptide, a process influenced by other matrix metalloproteinases (e.g., MMP2, MMP3, and MMP14), possibly involving multiple cleavage steps. Activation can also occur through cleavage by other proteases or spontaneous cleavage under 4-aminophenylmercuric acetate (APMA) treatment (in vitro).
• - N-glycosylation and phosphorylation are known post-translational modifications.

WB Experiment Tips

Precautions

• MMP13 is a protein secreted into the extracellular matrix or extracellular space. We recommend concentrating cell culture supernatants using ultrafiltration or ammonium sulfate precipitation before detection if no signal is detected.
• For specific cells like human chondrosarcoma cells, induction with IL-1β can enhance MMP13 expression, which helps address issues of weak or absent signals.
• MMP13 is synthesized as a pro-form (~60 kDa) and secreted into the extracellular space. Various cleavage fragments (~34 kDa, ~54 kDa) can be generated through processing. Therefore, multiple bands may appear in WB experiments.
• We recommend not cutting membranes and retaining the entire membrane for experiments.

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

• Add a complex proteinase inhibitor to avoid degradation of the target protein.
• Select an appropriate lysis buffer to enrich for more target protein.
• Keep the sample on ice throughout the sample preparation process.
• Determine the protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

• Load at least 20 μg total protein for electrophoresis.
• It is recommended to use positive and negative controls.

Transfer:

• After the PVDF membrane is activated, wash it thoroughly to remove residual methanol from the membrane completely.
• We recommend using Ponceau S staining after transfer to determine if the transfer is successful (if fluorescence labeling detection is selected, make sure Ponceau S is completely washed off).

Blocking:

• There is no blocking solution applicable to all systems. Please choose a suitable blocking solution.

Positive Controls

• HeLa cell lysate
• Concentrated cell culture supernatant from IL-1β-treated human chondrosarcoma cells
• Mouse chondrocyte lysate [5]
• Rat chondrocyte lysate [6,7]

Example Results

Figure 2: WB Experiment Results of MMP13 Protein, Anti-MMP13 Antibody (ab51072)

Lane 1: HeLa cell lysate

Loading amount: 15 μg
Primary antibody dilution: 1:1000
Predicted band size: 54 kDa
Observed band size: 60 kDa

Figure 3: WB Experiment Results of MMP13 Protein, Anti-MMP13 Antibody (ab39012)

Lane 1: Human chondrosarcoma cell (untreated) culture supernatant
Lane 2: Human chondrosarcoma cell (IL-1β treated) culture supernatant

Loading amount: Concentrated 40-fold, 15 μL/lane
Primary antibody dilution: 1:3000
Predicted band size: 54 kDa
Observed band size: 60 kDa

IHC Experiment Tips

Precautions

• MMP13 is expressed in mature osteoblasts, while other tissues are mostly negative under normal conditions. When conducting IHC experiments, we recommend using cortical bone as a positive control.
• When detecting MMP13 by IHC in highly mineralized bone tissue samples, after fixation, it is necessary to undergo decalcification to meet subsequent staining requirements. The decalcification time and choice of decalcifying agent usually need to be optimized based on tissue size, type, and bone density. During decalcification, it is essential to monitor the degree of decalcification continuously. Well-decalcified tissue should have a cartilage-like or rubbery texture, and the endpoint of decalcification can be assessed by needle penetration, where slight resistance indicates complete decalcification. For more detailed theories and practices in bone histology, refer to the cited reference [8].
• To prevent bone tissue sections from detaching during the experimental process, optimization can be achieved through the following measures: 1) use of poly-l-lysine-coated slides; 2) avoid overly thick sectioning (<5 µm); 3) select microwave antigen retrieval; 4) allow natural cooling after retrieval, avoiding sudden temperature drops; 5) gently rinse slides to avoid direct PBS flushing of sections; 6) after incubating with primary antibodies at 4°C overnight, gradually warm to room temperature.

Positive Controls

• Mouse/Rat Femoral Tissue

• Human Tibial Tissue

Example Results

Image 4: IHC-P experimental result of MMP13 protein using Anti-MMP13 antibody [EPR21778] (ab219620).

Sample name: Rat femur paraffin section.
Primary antibody: Diluted 2000 times.
Antigen retrieval method: Heat-induced antigen retrieval using Tris/EDTA buffer (pH 9.0)

Key control points

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

Sample fixation:

1. The sample fixation time depends on the size of the tissue block and the type of tissue, but for most samples, such as 4% PFA fixation, room temperature fixation for 18-24 hours is more appropriate.
2. Insufficient fixation will result in a higher signal at the edge of the sample and a weaker signal in the center, or even no signal.
3. Over-fixation will block the antigen epitope. Although antigen repair will expose some of the epitopes, if the tissue fixation time is very long (such as more than one week), there will be no signal after antigen repair.

Antigen repair:

1. When performing immunohistochemistry experiments on paraffin sections, we recommend using a pressure cooker for heat-induced antigen repair. You can try to repair the sections at 110℃ for 15 minutes. After repair, cool naturally and avoid putting them in cold water to prevent the temperature from dropping suddenly and causing the sections to fall off.
2. For tissues that are brittle and easy to peel off during high temperature and high pressure repair (such as bone, cartilage); or tissues with small sections, such as sciatic nerve, microwave repair can also be selected.

Blocking:

1. If HRP conjugates are used for subsequent detection, please use 3% hydrogen peroxide to treat the slices for 10 minutes to block endogenous peroxidase.
2. If a fluorescent group-conjugated secondary antibody is used for the experiment, it is recommended to use a blocking solution with 1% BSA and a final concentration of 0.3M glycine to quench the autofluorescence caused by aldehyde groups.

References

1. V Knäuper, C López-Otin, B Smith et al. Biochemical characterization of human collagenase-3. J Biol Chem. (1996);271(3):1544-50. doi: 10.1074/jbc.271.3.1544.
2. P Borden, D Solymar, A Sucharczuk et al. Cytokine control of interstitial collagenase and collagenase-3 gene expression in human chondrocytes. J Biol Chem. (1996); 271(38):23577-81. doi: 10.1074/jbc.271.38.23577.
3. N Johansson, U Saarialho-Kere, K Airola, R Herva et al. Collagenase-3 (MMP-13) is expressed by hypertrophic chondrocytes, periosteal cells, and osteoblasts during human fetal bone development. Dev Dyn. (1997) ; 208(3):387-97. doi: 10.1002/(SICI)1097-77(199703)208:3<387::AID-AJA9>3.0.CO;2-E.
4. Mattia R Bordoli, Jina Yum, Susanne B Breitkopf et al. A secreted tyrosine kinase acts in the extracellular environment. Cell. (2014) 158(5):1033-1044. doi: 10.1016/j.cell.2014.06.048.
5. Bo Yan, Zhongmin Zhang, Dadi Jin et al. mTORC1 regulates PTHrP to coordinate chondrocyte growth, proliferation and differentiation. Nat Commun. 2016 Apr 4;7: 11151.doi: 10.1038/ncomms11151.
6. Sheng-Long Ding, Zhi-Ying Pang, Xue-Mei Chen et al. Urolithin a attenuates IL-1β-induced inflammatory responses and cartilage degradation via inhibiting the MAPK/NF-κB signaling pathways in rat articular chondrocytes. J Inflamm (Lond). 2020 Mar 24;17:13. doi: 10.1186/s12950-020-00242-8. eCollection 2020.
7. Guang Yang, Siying Li, Bin Li et al. Protective effects of garlic-derived s-allylmercaptocysteine on IL-1β-stimulated chondrocytes by regulation of MMPs/TIMP-1 Ratio and Type II Collagen expression via suppression of NF-κB pathway. Biomed Res Int. 2017;2017:8686207. doi: 10.1155/2017/8686207. Epub 2017 Dec 3.
8. Gayle M. Callis,18 - Bone, Editor(s): John D. Bancroft, Marilyn Gamble, Theory and Practice of Histological Techniques (Sixth Edition),Churchill Livingstone,2008,Pages: 333-363,https://doi.org/10.1016/B978-0-443-10279-0.50025-7.