MMP23A Antibody

What is MMP23A and why is it targeted in research applications?

MMP23A (Matrix Metalloproteinase 23A) is a zinc- and calcium-dependent endopeptidase that functions in extracellular matrix organization and proteolysis. The protein is encoded by the MMP23B gene (with MMP23A and MMP23B being identical genes on chromosome 1). It has a canonical length of 390 amino acid residues and a molecular mass of 43.9 kDa . MMP23 is primarily localized in the endoplasmic reticulum and shows high expression in reproductive tissues including ovary, testis, and prostate . Its involvement in matrix remodeling, enchondral bone formation, and regulation of cranial suture closure makes it a target of interest in developmental biology, reproductive studies, and cancer research .

What are the key structural domains of MMP23A that antibodies may target?

MMP23 has a unique domain structure compared to other MMPs:

MMP23 is distinct from other MMPs due to its unique domain architecture, making domain-specific antibodies particularly valuable for studying its various functions .

What are the common alias names for MMP23A in research literature?

When searching for MMP23A antibodies in literature, researchers should be aware of multiple aliases:

• MIFR, MIFR-1 (early nomenclature)

• MMP22, MMP21 (alternative designations)

• MMP23B (identical gene duplicate)

• Femalysin (functional description in reproductive tract)

• Matrix metalloproteinase in the female reproductive tract

This diversity in nomenclature requires careful literature searching to ensure all relevant research is identified.

What are the most validated applications for MMP23A antibodies?

Based on commercial antibody validation data, MMP23A antibodies are primarily validated for:

Immunofluorescence is the most common application for MMP23A antibodies, followed by ELISA and Western Blot .

How should tissue preparation and antigen retrieval be optimized for MMP23A immunohistochemistry?

For optimal MMP23A detection in formalin-fixed, paraffin-embedded tissues:

• After deparaffinization and rehydration, perform heat-induced epitope retrieval in 0.01M citrate buffer, pH 6.0, for 20 minutes at high power in a 1,200-watt microwave .

• Cool sections in tap water for 5 minutes.

• Quench with 0.3% hydrogen peroxide for 30 minutes.

• Block with diluted normal serum (goat serum works well for rabbit primary antibodies).

• Incubate with MMP23A primary antibody at 1:100 dilution at room temperature for 1 hour, then at 4°C overnight.

• Continue with appropriate secondary antibody (e.g., biotinylated goat anti-rabbit at 1:500) and detection system.

This protocol has been validated for robust MMP23A detection in melanoma tissues and can be adapted for other tissue types .

What controls should be included when using MMP23A antibodies in research?

When designing experiments with MMP23A antibodies, include these essential controls:

• Positive tissue controls: Placenta, ovary, testis, and prostate tissues have been validated as positive controls for MMP23A expression .

• Antibody specificity controls:

  • Peptide competition assay: Pre-incubate MMP23A antibody (1 μg/ml) with immunizing peptide (2 μg/ml) for 1 hour before application to verify specificity .

  • Western blot verification: Run protein extracts from known MMP23A-expressing tissues alongside experimental samples .

• Negative controls: Include isotype-matched non-specific antibody controls to assess background staining.

• siRNA knockdown controls: Where possible, include MMP23A siRNA-treated samples to confirm antibody specificity to the target protein .

These controls are essential for validating experimental results and ensuring antibody specificity .

How can I address weak or absent signal when using MMP23A antibodies in Western blotting?

When troubleshooting weak MMP23A Western blot signals:

• Sample preparation optimization:

  • Ensure sufficient protein is loaded (≥10 μg for tissue extracts) .

  • Use a lysis buffer containing adequate protease inhibitors to prevent MMP23A degradation.

  • Consider enriching ER membrane fractions, as MMP23A is primarily localized to the ER .

• Antibody selection and optimization:

  • Test antibodies targeting different epitopes (N-terminal, C-terminal, or internal regions).

  • C-terminal antibodies (such as those targeting AA 341-390) often show better detection in Western blot applications .

  • Optimize primary antibody concentration (start with 1:500 dilution and adjust as needed) .

• Detection enhancement:

  • Consider using high-sensitivity chemiluminescent substrates.

  • Extend primary antibody incubation time (overnight at 4°C).

  • Use fresh antibody to avoid degradation issues.

• Technical considerations:

  • Ensure adequate transfer of high-molecular-weight proteins.

  • Consider native vs. reducing conditions based on the antibody's specifications.

These approaches address the common challenges in detecting MMP23A in Western blotting applications .

What strategies can improve specificity when using MMP23A antibodies for immunohistochemistry?

To enhance specificity in MMP23A immunohistochemistry:

• Antibody selection: Choose antibodies validated specifically for IHC applications, as not all MMP23A antibodies perform equally across applications .

• Optimization steps:

  • Test multiple antigen retrieval methods (citrate-based vs. EDTA-based).

  • Perform titration experiments to determine optimal antibody concentration (typically 1:25-1:100) .

  • Extend primary antibody incubation time (overnight at 4°C).

• Background reduction:

  • Use appropriate blocking sera (goat serum for rabbit primaries).

  • Include 0.1% Triton X-100 in blocking solutions to reduce non-specific binding.

  • Consider avidin-biotin blocking if using biotin-based detection systems.

• Validation approaches:

  • Perform peptide competition assays to confirm specificity.

  • Compare staining patterns with mRNA expression data.

  • Implement multi-antibody approach using antibodies targeting different epitopes.

These techniques have been successfully employed to achieve specific MMP23A staining in various tissue types, including melanoma specimens .

How do I distinguish between MMP23A and related MMPs when using antibodies in multi-marker studies?

When conducting research requiring discrimination between MMP23A and other MMPs:

• Antibody selection criteria:

  • Prioritize antibodies raised against unique regions of MMP23A not conserved in other MMPs.

  • Antibodies targeting the C-terminal region (AA 341-390) show high specificity for MMP23A with minimal cross-reactivity .

  • Verify the manufacturer's cross-reactivity testing data against other MMPs, particularly closely related family members.

• Experimental validation:

  • Perform Western blots with recombinant MMP proteins to confirm specificity.

  • Include siRNA knockdown controls specific to MMP23A.

  • Consider dual staining with antibodies against different MMP23A epitopes to confirm specificity.

• Data interpretation considerations:

  • Be aware that some commercially available antibodies may cross-react with both MMP23A and MMP23B due to their high sequence homology.

  • When analyzing expression in tissues with known expression of multiple MMPs (such as inflamed tissues), use additional techniques (qPCR, in situ hybridization) to confirm antibody specificity.

These approaches help ensure reliable discrimination between MMP23A and related matrix metalloproteases in complex experimental systems .

What scoring systems are recommended for quantifying MMP23A expression in immunohistochemistry?

For standardized quantification of MMP23A expression in tissue samples, implement this validated scoring approach:

• Composite scoring system:

  • Staining intensity: 0 (none), 1 (faint), 2 (intense), 3 (very intense)

  • Distribution score: 0 (none), 1 (focal, <50%), 2 (diffuse, ≥50%)

  • Calculate composite score by summing intensity and distribution scores (range: 0-5)

• Field selection methodology:

  • Scan each slide at 100x magnification

  • Identify the field with highest antibody expression

  • Score a representative high-power field (0.2 mm²)

• Blinded assessment:

  • Have scoring performed by a pathologist blinded to clinical data

  • For research studies, have multiple observers score independently

This scoring system has been validated in melanoma research and allows for statistical comparison between different patient groups or experimental conditions .

How should I analyze MMP23A expression in relation to clinical parameters in cancer studies?

When analyzing MMP23A expression in cancer research:

These analytical approaches have revealed meaningful clinical correlations in melanoma research, where high MMP23A expression was associated with decreased tumor-infiltrating lymphocytes, increased regulatory T cells, and poorer response to immune biologics .

How do alternative splice variants of MMP23 affect antibody detection and data interpretation?

When interpreting MMP23A antibody results, consider splice variant complexity:

• Known splice variants:

  • MMP23B has three reported isoforms produced by alternative splicing with molecular weights of 44 kDa, 46 kDa, and 48 kDa

  • These variants may affect epitope availability and antibody detection

• Antibody detection considerations:

  • Western blots may show multiple bands corresponding to different splice variants

  • Verify which isoforms your antibody is expected to detect based on the epitope location

  • Consider using multiple antibodies targeting different regions to detect all relevant isoforms

• Analytical approaches:

  • Document all observed bands in Western blot experiments

  • When possible, identify which specific isoforms are being detected

  • In tissues with complex expression patterns, consider complementary mRNA analysis to identify specific transcript variants

• Reporting standards:

  • Explicitly state which MMP23 isoforms were detected in methods and results sections

  • Include representative images showing all detected bands/isoforms

  • Discuss limitations in discriminating between closely related isoforms

This understanding of splice variant complexity is essential for accurate interpretation of MMP23A antibody data and meaningful cross-study comparisons .

How can MMP23A antibodies be used to investigate its role in immune modulation via Kv1.3 channel interactions?

Advanced researchers investigating MMP23A's immunomodulatory functions through Kv1.3 channel interactions should consider:

• Co-localization studies:

  • Use dual immunofluorescence with anti-MMP23A and anti-Kv1.3 antibodies

  • Employ high-resolution microscopy (confocal or super-resolution) to assess physical proximity

  • Include appropriate co-localization controls and quantification metrics

• Functional interaction assessment:

  • Combine MMP23A antibody staining with patch-clamp electrophysiology to correlate expression with channel function

  • Use proximity ligation assays to detect direct protein-protein interactions between MMP23A and Kv1.3

  • Consider live-cell imaging with fluorescently tagged antibody fragments to track dynamic interactions

• Mechanistic investigation approaches:

  • Use domain-specific antibodies to identify which MMP23A domains interact with Kv1.3

  • Apply blocking antibodies against specific MMP23A domains to disrupt Kv1.3 interactions

  • Combine with MMP23A siRNA knockdown to validate antibody-based findings

These advanced approaches have revealed that MMP23 can regulate immune responses by blocking voltage-gated potassium channel Kv1.3, whose function is important for sustained Ca2+ signaling during T cell activation .

What are the most effective protocols for using MMP23A antibodies in studying cancer immunomodulation?

For investigating MMP23A's role in tumor immune evasion:

This approach has revealed that high melanoma MMP23 expression is associated with recurrence in patients treated with immune biologics and shorter periods of progression-free survival, suggesting potential as both a predictive biomarker and therapeutic target .

How can researchers employ MMP23A antibodies to study its involvement in inflammatory bowel disease and other inflammatory conditions?

For investigating MMP23A's role in inflammatory conditions:

• Comparative expression analysis:

  • Use standardized IHC protocols to compare MMP23A expression in:

    • Active inflammation vs. remission tissues

    • Different inflammatory conditions (e.g., UC vs. CD in IBD)

    • Inflamed vs. adjacent normal tissue

• Cell-specific expression profiling:

  • Employ MMP23A antibodies in multi-color flow cytometry to identify expressing cell populations

  • Use laser capture microdissection combined with immunostaining to isolate specific cell populations

  • Perform single-cell analysis with MMP23A antibodies to reveal heterogeneity in expression

• Pathway integration analysis:

  • Combine MMP23A antibody staining with markers of:

    • EMMPRIN (CD147, an MMP inducer)

    • Other MMPs potentially coregulated (MMP10)

    • NF-κB pathway components

    • Wnt/β-catenin signaling molecules

These approaches have revealed that MMP23 levels are increased in active ulcerative colitis and Crohn's disease compared to remission states and control groups, suggesting involvement in the inflammatory process . Similar methodologies can be applied to study MMP23A's role in other inflammatory conditions.