MMP14 Antibody

What is MMP14 and why is it significant in research?

MMP14, also known as MT1-MMP (Membrane Type 1 Matrix Metalloproteinase), is a cell membrane-bound proteinase that plays a crucial role in tumor growth, invasion, and metastasis. It enhances degradation of collagen IV, a major component of the basement membrane, by forming a complex with tissue inhibitor of metalloproteinase-2 (TIMP-2) to activate pro-MMP-2. MMP14 influences venous invasion, intrahepatic metastasis, and patient outcomes in hepatocellular carcinoma (HCC) . Additionally, research has shown that MMP14 may be present in the centromere and could lead to chromosome instability, suggesting potential novel functions in intracellular compartments beyond its well-established extracellular roles .

What forms of MMP14 can be detected by antibodies?

Most validated MMP14 antibodies can detect multiple forms of the protein. In western blotting, pro-MMP14 (65 kDa) and active MMP14 (51 kDa) bands can be detected along with truncated MMP14 forms (45, 42, 35, 20 kDa) . The observed molecular weight of MMP14 is typically reported as 66 kDa, with some antibodies also detecting the 45-50 kDa processed forms . The choice of antibody should be based on which form(s) of MMP14 are relevant to your specific research question and experimental system.

What species reactivity is available for MMP14 antibodies?

Most commercially available MMP14 antibodies demonstrate reactivity with human, mouse, and rat samples . Some antibodies like AF0212 are predicted to also recognize MMP14 from additional species such as pig, bovine, horse, sheep, rabbit, and dog . When selecting an antibody for cross-species applications, it is advisable to verify the homology of the target epitope and conduct preliminary validation tests in your specific experimental system.

What are the recommended applications and dilutions for MMP14 antibodies?

MMP14 antibodies can be utilized in multiple applications with specific recommended dilutions:

It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as optimal dilutions may be sample-dependent . Published literature demonstrates successful use of MMP14 antibodies in WB (22+ publications), IHC (7+ publications), and IF (5+ publications) as documented for antibody 14552-1-AP .

How can I validate MMP14 antibody specificity for my experiments?

Validating MMP14 antibody specificity requires a multi-faceted approach:

• Blocking peptide validation: Use a specific blocking peptide containing the immunogen sequence to confirm antibody specificity.

• Independent antibody verification: Compare results using two antibodies targeting different epitopes of MMP14.

• Phospho-specificity validation: If studying phosphorylated forms, verify specificity using phosphatase treatment.

• Genetic validation: Use MMP14 knockout/knockdown cells as negative controls.

• Cell line validation: Confirm expression patterns in cell lines known to express varying levels of MMP14. Published data shows positive WB detection in A549 cells and human colon tissue, and positive IF/ICC in HepG2 and COLO 320 cells .

The most rigorous approach combines multiple validation methods to ensure antibody specificity in your particular experimental system.

How can MMP14 antibodies be used to investigate cancer stem cell populations?

Recent research has revealed that cancer stem cells express significantly higher levels of MMP14 compared to bulk tumor cells. Flow cytometry studies using MMP14 antibodies have demonstrated that CD29+/ALDH+ cancer stem cell populations express approximately 2-fold higher MMP14 mRNA levels than bulk tumor cells . Specifically, around 4.0±1.8% of CD29+ALDH+ cells stained positively for MMP14, while less than 0.1% of bulk cells were MMP14+ .

To study MMP14 expression in cancer stem cells:

• Isolate stem cell populations using established markers (e.g., CD29, ALDH activity)

• Perform flow cytometry using MMP14 antibodies with appropriate controls

• Compare expression levels between stem and non-stem populations

• Use functional assays (such as sphere-formation) with and without MMP14 inhibitory antibodies to assess the role of MMP14 in stem cell function

This approach can provide insights into the role of MMP14 in cancer stem cell biology, metastasis initiation, and therapeutic resistance mechanisms.

What are the considerations for developing inhibitory antibodies against MMP14?

Developing inhibitory antibodies against MMP14 presents unique challenges due to the concave structure of its catalytic site. Conventional antibodies typically have flat or concave paratopes that are incompatible with binding to the active site of enzymes like MMP14 . To overcome this limitation, researchers have developed specialized approaches:

• Extended CDR-H3 strategies: Synthetic antibody libraries carrying extended, 23- to 27-residue, complementarity-determining region (CDR)–H3 segments have been developed to create convex paratopes that can access the active site .

• Camelid-inspired designs: Human antibodies inspired by camelid immunoglobulins with convex-shaped paratopes have proven effective at inhibiting MMP14 activity .

• Selectivity assessment: Rigorous testing against multiple MMPs is crucial, as many previous MMP inhibitors failed due to broad-specificity rather than selective inhibition .

From these approaches, antibodies like Fab 3A2 have demonstrated potent and selective inhibition of MMP14 without cross-reactivity to other MMPs . The IgG form of 3A2 has shown therapeutic potential in breast cancer models, reducing metastatic spread to the lungs and liver by 94% .

How can MMP14 antibodies be used to study the different cellular localizations of MMP14?

MMP14 has been detected in multiple cellular locations beyond its classical membrane localization, including the centromere, suggesting diverse functions . To investigate these varied localizations:

• Subcellular fractionation: Perform fractionation followed by western blotting with MMP14 antibodies to detect differential distribution.

• Co-localization studies: Use dual immunofluorescence with MMP14 antibodies and markers for different cellular compartments (membrane, cytoskeletal, nuclear, centrosomal).

• Super-resolution microscopy: Employ techniques like STORM or STED with specific MMP14 antibodies to precisely determine subcellular localization.

• Live-cell imaging: Use fluorescently-tagged anti-MMP14 antibody fragments (if cell-permeable) or fluorescently-tagged MMP14 to track localization changes during cellular processes.

These approaches can help elucidate the non-canonical functions of MMP14 in different cellular compartments and potentially identify novel therapeutic strategies.

Why might I observe multiple MMP14 bands in Western blotting?

The observation of multiple bands in MMP14 Western blotting is common and represents different forms of the protein:

• Pro-MMP14 (65-66 kDa): The inactive zymogen form with the pro-domain intact.

• Active MMP14 (51-55 kDa): The mature, catalytically active form after pro-domain removal.

• Truncated forms (45, 42, 35, 20 kDa): Results of autocatalytic processing or proteolytic cleavage by other proteases .

If unexpected bands appear, consider:

• Confirming specificity with blocking peptides

• Using positive and negative control cell lines

• Optimizing sample preparation to minimize degradation

• Verifying that extraction methods are suitable for membrane proteins

• Comparing results with different antibodies targeting distinct epitopes

The observed pattern of bands can provide valuable information about MMP14 processing and activation states in your experimental system.

What are optimal fixation and sample preparation methods for MMP14 immunostaining?

For optimal MMP14 immunostaining results:

For cell lines (IF/ICC):

• Fixation: 4% paraformaldehyde (10-15 min) preserves structure while maintaining antigenicity.

• Permeabilization: 0.1-0.2% Triton X-100 (10 min) for balanced access to membrane and intracellular MMP14.

• Blocking: 5% BSA or 10% normal serum (1 hour) to minimize non-specific binding.

• Antibody incubation: Follow recommended dilutions (typically 1:200-1:800) , and incubate overnight at 4°C.

For tissue sections (IHC):

• Fixation: 10% neutral buffered formalin, followed by paraffin embedding.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

• Blocking endogenous peroxidase: 3% H₂O₂ treatment if using HRP detection.

• Primary antibody incubation: Overnight at 4°C with validated MMP14 antibodies at specified dilutions.

Researchers should optimize these conditions for their specific samples, as expression levels and accessibility of MMP14 can vary across different cell types and tissues.

How can MMP14 antibodies be used to distinguish between active and inactive forms in complex biological samples?

Distinguishing between active and inactive MMP14 forms requires specialized approaches:

• Active-site specific antibodies: Some engineered antibodies like Fab 3A2 specifically recognize and bind to the active site, only interacting with the active form .

• Conformation-specific antibodies: Antibodies that recognize structural differences between pro-MMP14 and active MMP14.

• Functional assays: Combining immunoprecipitation with MMP14 antibodies followed by activity assays against known substrates.

• Proximity ligation assays: To detect MMP14 interactions with TIMP-2 or proMMP-2, which indicate the active complex formation.

• Zymography following immunoprecipitation: To assess the activity of MMP14 after isolation with specific antibodies.

These approaches enable researchers to investigate the activation status of MMP14 in various physiological and pathological contexts, providing insights into its regulation and function.

What are the emerging therapeutic applications of inhibitory MMP14 antibodies?

Inhibitory antibodies against MMP14 show promising therapeutic potential in several disease contexts:

• Cancer metastasis inhibition: IgG 3A2, a selective MMP14 inhibitory antibody, has demonstrated remarkable efficacy in reducing metastatic spread to the lungs and liver by 94% in a highly metastatic breast cancer model .

• Cancer stem cell targeting: MMP14 inhibitory antibodies can suppress the sphere-forming capacity and proliferation of cancer stem cells, potentially addressing the challenge of metastatic relapse .

• Selective targeting: Unlike broad-spectrum MMP inhibitors that failed in clinical trials due to off-target effects, selective antibodies like Fab 3A2 specifically inhibit MMP14 without affecting other MMPs .

• Combinatorial approaches: Current research is exploring combinations of MMP14 inhibitory antibodies with established cancer therapies to enhance efficacy.

The development pipeline established for creating inhibitory antibodies targeting MMP14 can be applied to generate inhibitors for other enzymes, expanding the therapeutic potential beyond MMPs alone .