Cleaved-MMP10 (F99) Antibody

What is Cleaved-MMP10 (F99) Antibody and what does it detect?

Cleaved-MMP10 (F99) Antibody is a research-grade antibody specifically designed to detect endogenous levels of activated MMP-10 protein fragments resulting from cleavage adjacent to the F99 residue. It's available in both polyclonal (rabbit) and monoclonal (mouse) formats. The antibody recognizes a synthetic peptide derived from the internal region (AA range 80-129) of human MMP-10 . This specificity makes it valuable for studying MMP-10 activation states in various physiological and pathological contexts, particularly in extracellular matrix remodeling processes.

What is the target protein and its physiological significance?

Matrix metalloproteinase-10 (MMP-10, also known as Stromelysin-2 or SL-2) is a secreted protease involved in the breakdown of extracellular matrix components. The protein plays crucial roles in normal physiological processes including embryonic development, reproduction, and tissue remodeling, as well as in pathological conditions such as arthritis and cancer metastasis . Functionally, MMP-10 can degrade fibronectin, multiple types of gelatins (types I, III, IV, and V), and to a lesser extent collagens III, IV, and V. It also activates procollagenase, functioning as part of the complex metalloproteinase activation cascade .

What are the biochemical properties of the antibody and target protein?

The Cleaved-MMP10 (F99) polyclonal antibody is purified by affinity chromatography using epitope-specific immunogen . The target protein MMP-10 has a molecular weight of approximately 54 kDa by SDS-PAGE analysis, though the cleaved form detected by this specific antibody appears at approximately 43 kDa on Western blots . MMP-10 contains a conserved cysteine in the cysteine-switch motif that binds catalytic zinc ions, inhibiting enzyme activity until the activation peptide is released . The protein contains four hemopexin-like domains and belongs to the peptidase M10A family .

What are the validated applications for this antibody?

The Cleaved-MMP10 (F99) Antibody has been validated for Western blot (WB) and ELISA applications . For Western blotting, the recommended dilution range is 1:500-1:2000, while for ELISA applications, a higher dilution of 1:40000 may be used . Although immunohistochemistry applications are not explicitly validated in the provided search results, the positive reactivity in tissues such as coronary artery and ovary suggests potential utility in tissue-based detection methods .

How should sample preparation be optimized for detecting cleaved MMP-10?

For optimal detection of cleaved MMP-10, researchers should consider the following sample preparation guidelines:

• Cell/tissue lysis: Use a buffer containing protease inhibitors to prevent artificial activation during processing

• Subcellular fractionation: Since MMP-10 is secreted to the extracellular space , analyze both cellular and media/extracellular fractions

• Denaturation: Complete protein denaturation is critical for exposing the cleaved epitope

• Loading controls: Include appropriate loading controls for secreted proteins rather than typical housekeeping proteins

• Positive controls: Consider using coronary artery or ovary tissue samples as positive controls

For optimal Western blot results, ensure samples are prepared under reducing conditions and heat-denatured appropriately before loading.

What are the recommended storage and handling conditions for maintaining antibody activity?

For optimal preservation of antibody activity, store the Cleaved-MMP10 (F99) Antibody at -20°C for up to one year . After initial thawing, the antibody can be kept at +4°C for short-term use. For long-term storage, it is recommended to prepare aliquots and store them at either -20°C or -80°C to avoid repeated freeze-thaw cycles that may compromise antibody performance . The antibody is formulated in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide as a preservative .

What factors might affect specificity when detecting cleaved MMP-10?

Several factors can impact the specificity of cleaved MMP-10 detection:

• Cross-reactivity with related MMPs: Due to structural similarities among matrix metalloproteinases, especially MMP-3 (stromelysin-1)

• Sample processing artifacts: Inadvertent activation during sample preparation

• Species-specific variations: Although the antibody is reactive with human MMP-10, cross-reactivity with rodent orthologs may vary between polyclonal and monoclonal versions

• Protease inhibitors: Absence of appropriate inhibitors during sample preparation may lead to artificial activation patterns

• Background signals: Non-specific binding in complex biological samples

To ensure specificity, researchers should include appropriate positive and negative controls and validate results using complementary methods.

How can I differentiate between latent and cleaved forms of MMP-10?

Distinguishing between latent (pro-MMP-10) and cleaved (active) forms requires careful experimental design:

• Molecular weight discrimination: The latent form appears at approximately 54 kDa while the cleaved form appears at approximately 43 kDa on Western blots

• Use of specific antibodies: The Cleaved-MMP10 (F99) Antibody specifically detects fragments resulting from cleavage adjacent to F99, whereas antibodies recognizing total MMP-10 will detect both forms

• Activity assays: Complement immunodetection with functional activity assays

• Sequential immunoprecipitation: Use antibodies against different epitopes to separate latent and cleaved forms

• Comparison with known activators: Include samples treated with activators like APMA (4-aminophenylmercuric acetate) to generate reference patterns

A side-by-side comparison of samples on the same blot using both total MMP-10 and cleaved-specific antibodies provides the most reliable differentiation.

What are common pitfalls in Western blot detection of cleaved MMP-10?

Researchers should be aware of these common challenges when performing Western blot for cleaved MMP-10:

• Insufficient denaturation: Incomplete denaturation may mask the epitope

• Inappropriate blocking: Overly stringent blocking can reduce specific signal

• Cross-reactivity with other MMPs: Particularly with MMP-3, which shares structural similarity

• Low abundance in some samples: May require concentration of culture media or extracellular fractions

• Activation during sample processing: Can artificially increase the cleaved form signal

• Migration pattern variations: Different electrophoresis systems may show slight variations in apparent molecular weight

For optimal results, use freshly prepared samples with appropriate protease inhibitors and follow the recommended antibody dilutions (1:500-1:2000) .

How can the Cleaved-MMP10 (F99) Antibody be utilized in studying MMP activation cascades?

The antibody can serve as a valuable tool in investigating MMP activation networks through several approaches:

• Temporal activation patterns: Monitor cleaved MMP-10 appearance following stimulation with known MMP activators

• Inhibitor studies: Assess the effect of various protease inhibitors on MMP-10 activation

• Co-immunoprecipitation: Identify protein complexes involved in activation

• Correlation with substrate degradation: Connect MMP-10 activation with degradation of specific matrix components

• Activation in disease models: Compare activation patterns between normal and pathological conditions

Since MMP-10 can activate procollagenase , studying its cleaved form provides insight into amplification mechanisms within the metalloproteinase cascade.

What experimental approaches can distinguish MMP-10 from other stromelysins in complex samples?

Distinguishing MMP-10 (stromelysin-2) from related stromelysins, particularly MMP-3 (stromelysin-1), requires specialized approaches:

• Epitope-specific antibodies: The Cleaved-MMP10 (F99) Antibody targets a specific region (AA 80-129) that may differ from homologous regions in other stromelysins

• Sequential immunodepletion: Remove one stromelysin subtype and analyze the remaining signal

• Mass spectrometry validation: Confirm immunoblot findings with peptide mass fingerprinting

• Substrate specificity assays: Exploit subtle differences in substrate preferences

• Knockout/knockdown validation: Verify signal specificity using genetic manipulation approaches

A combination of these approaches provides the most robust distinction between closely related stromelysin family members.

How can this antibody contribute to studying matrix remodeling in disease models?

The Cleaved-MMP10 (F99) Antibody offers several advantages for investigating extracellular matrix dynamics in pathological conditions:

• Biomarker potential: Monitor activation status as a potential marker of disease progression

• Intervention assessment: Evaluate therapeutic strategies targeting matrix remodeling

• Compartment-specific analysis: Compare activation patterns in different tissue compartments

• Correlation with clinical parameters: Link MMP-10 activation with disease severity indices

• Microenvironment studies: Investigate stromal-epithelial interactions mediated by active MMP-10

Since MMP-10 is involved in disease processes such as arthritis and cancer metastasis , detecting its activated form can provide mechanistic insights into pathological matrix degradation events.

How should I interpret differences in MMP-10 activation patterns between experimental conditions?

When analyzing variations in MMP-10 activation across different experimental conditions, consider these interpretative frameworks:

• Ratio analysis: Calculate the proportion of cleaved to total MMP-10 as an activation index

• Temporal dynamics: Assess not just the magnitude but also the kinetics of activation

• Correlation with outcomes: Connect activation patterns with functional endpoints (e.g., invasion, migration)

• Context-dependent activation: Interpret results within the specific tissue/cellular microenvironment

• Validation with activity assays: Confirm that immunologically detected cleaved forms correlate with enzymatic activity

Remember that changes in cleaved MMP-10 levels may reflect alterations in either the rate of proenzyme activation or the stability/clearance of the active form.

What controls are essential for validating cleaved MMP-10 detection in research studies?

A robust experimental design for cleaved MMP-10 detection should incorporate these controls:

• Positive tissue controls: Coronary artery or ovary tissues known to express MMP-10

• Activation controls: Samples with chemically induced activation (e.g., APMA treatment)

• Inhibition controls: Samples treated with MMP inhibitors to prevent activation

• Migration standards: Molecular weight markers to confirm the 43 kDa band corresponds to the cleaved form

• Specificity controls: Preabsorption with the immunizing peptide to verify signal specificity

• Cross-method validation: Confirmation of results using alternative detection methods

Implementation of these controls ensures that observed signals genuinely represent the cleaved MMP-10 form and are not artifacts or misidentified related proteins.

How can researchers quantitatively assess MMP-10 activation in comparative studies?

For rigorous quantitative analysis of MMP-10 activation across experimental groups:

• Densitometric analysis: Standardized quantification of Western blot bands at 43 kDa (cleaved) relative to 54 kDa (pro-form)

• ELISA standard curves: Development of calibrated assays using recombinant standards

• Internal reference normalization: Use of consistent loading controls appropriate for secreted proteins

• Multiple biological replicates: Statistical power through adequate sample size

• Image analysis software: Utilize specialized software with background subtraction capabilities

• Activity-to-protein ratios: Correlate immunodetection with functional enzymatic activity

For the most meaningful comparisons, analyze samples from different experimental groups simultaneously on the same blot or plate to minimize technical variation.