Cleaved-MMP27 (Y99) Antibody

What is Cleaved-MMP27 (Y99) Antibody and what does it detect specifically?

The Cleaved-MMP27 (Y99) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of activated MMP-27 protein fragments resulting from cleavage adjacent to tyrosine 99 (Y99) . Matrix metalloproteinase-27 (MMP27) belongs to the MMP family involved in the breakdown of extracellular matrix components during normal physiological processes such as embryonic development, reproduction, and tissue remodeling . This antibody recognizes a specific epitope in the 80-129 amino acid range of human MMP27, making it valuable for studying MMP27 activation mechanisms .

What validated applications can Cleaved-MMP27 (Y99) Antibody be used for?

The Cleaved-MMP27 (Y99) Antibody has been validated primarily for:

Most suppliers have not extensively tested this antibody for other applications such as immunohistochemistry (IHC), immunofluorescence (IF), or immunoprecipitation (IP) . When exploring new applications, preliminary optimization experiments are highly recommended.

What is the optimal storage protocol for maintaining Cleaved-MMP27 (Y99) Antibody activity?

For long-term storage, keep the antibody at -20°C for up to one year from the date of receipt . For frequent use and short-term storage (up to one month), the antibody can be stored at 4°C . The antibody is typically supplied as a liquid in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . To maintain antibody integrity:

• Avoid repeated freeze-thaw cycles

• Consider aliquoting the antibody upon receipt

• Ensure proper temperature monitoring during storage

• Keep away from direct light exposure

How can I confirm the specificity of Cleaved-MMP27 (Y99) Antibody in my experiments?

To confirm antibody specificity:

• Run a blocking peptide control experiment using the synthesized immunogen peptide (amino acids 80-129 of human MMP27)

• Include positive control samples known to express cleaved MMP27 (such as COS7 cells treated with etoposide)

• Include a negative control sample where MMP27 is not expressed or activated

• Verify the detected band is at the expected molecular weight (~48 kDa for the cleaved form)

• Perform side-by-side comparisons with other validated MMP27 antibodies if available

What are the optimal experimental conditions for using Cleaved-MMP27 (Y99) Antibody in Western Blot analysis?

For optimal Western Blot results:

The antibody should detect a band of approximately 48 kDa representing the cleaved form of MMP27 . If working with monkey tissues, similar conditions apply, though optimization may be required .

How does MMP27 cleavage at Y99 affect its function and localization?

MMP27 is initially produced as an inactive proenzyme that requires proteolytic processing for activation. Cleavage adjacent to Y99 is a critical activation step that:

• Removes the inhibitory pro-domain, allowing substrate access to the catalytic domain

• Changes the protein's conformation, exposing the active site

• May influence its subcellular localization

While intact MMP27 is predominantly retained in the endoplasmic reticulum due to its C-terminal extension , the cleaved/activated form may have different trafficking patterns. The cleaved MMP27 functions in degrading extracellular matrix components including fibronectin, laminin, gelatins, and collagens . The Y99 cleavage site is strategically located to facilitate this activation process, making this antibody valuable for studying MMP27 processing and function in various physiological and pathological contexts.

What are the common technical challenges when using Cleaved-MMP27 (Y99) Antibody and how can they be addressed?

For weak signals specifically, consider using signal enhancement systems or more sensitive detection methods like fluorescent secondary antibodies for quantitative Western blot applications.

How can I use Cleaved-MMP27 (Y99) Antibody to study MMP27 activation in disease models?

For studying MMP27 activation in disease models:

• Comparative analysis: Compare MMP27 cleavage patterns between normal and diseased tissues or cell lines to identify differential activation.

• Time-course experiments: Monitor MMP27 activation during disease progression by collecting samples at different time points.

• Drug response studies: Evaluate how therapeutic compounds affect MMP27 activation by treating samples with drugs before analysis.

• Co-localization studies: Though this antibody is not validated for IF, if optimized, it could potentially be used to examine the spatial relationship between cleaved MMP27 and other proteins of interest.

• Activation triggers: Investigate factors that induce MMP27 cleavage in your model system by treating cells with cytokines, growth factors, or stress inducers.

This antibody is particularly valuable for studying MMP27's role in extracellular matrix remodeling in contexts like tissue development, wound healing, and cancer progression .

What controls should be included when using Cleaved-MMP27 (Y99) Antibody for research applications?

A robust experimental design should include:

• Positive control: Cell lines known to express cleaved MMP27 (COS7 cells treated with etoposide)

• Negative control:

  • Samples where MMP27 is not expressed

  • Primary antibody omission control to check for non-specific binding of secondary antibody

• Blocking peptide control: Preincubate the antibody with excess immunizing peptide (80-129 aa sequence) to demonstrate binding specificity

• Loading control: Use antibodies against housekeeping proteins like β-actin or GAPDH to ensure equal loading

• MMP family controls: If studying specificity within the MMP family, consider running samples expressing other MMPs to check for cross-reactivity

What sample preparation protocols maximize detection of cleaved MMP27?

For optimal detection of cleaved MMP27:

• Cell lysis buffer composition:

  • Use RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS)

  • Supplement with protease inhibitor cocktail to prevent further degradation

  • Include phosphatase inhibitors if studying phosphorylation events

• Sample handling:

  • Keep samples on ice during preparation

  • Process tissues quickly after collection to prevent degradation

  • Snap-freeze tissues in liquid nitrogen before homogenization

• Activation treatments:

  • Consider treating cells with activators like etoposide (25μM for 1h) to induce MMP27 cleavage

  • Other potential activators include phorbol esters, cytokines, or growth factors

• Protein denaturation:

  • Heat samples at 95°C for 5 minutes in Laemmli buffer

  • For membrane proteins, avoid excessive heating which may cause aggregation

How can I use this antibody to differentiate between the pro-form and cleaved form of MMP27?

To differentiate between MMP27 forms:

• Molecular weight analysis: The pro-form of MMP27 has a higher molecular weight than the cleaved form. Run both forms side-by-side on Western blots and compare with molecular weight markers. The cleaved form detected by this antibody appears at approximately 48 kDa .

• Induction experiments: Treat cells with agents known to induce MMP activation (e.g., etoposide) and compare treated vs. untreated samples .

• Sequential probing: Strip and reprobe membranes with antibodies recognizing different regions of MMP27 (pro-domain vs. catalytic domain) to identify the various forms.

• Enzymatic activity correlation: Correlate Western blot results with functional enzymatic assays to confirm that the cleaved form detected by the antibody corresponds to active MMP27.

• Time-course activation studies: Monitor the disappearance of the pro-form and appearance of the cleaved form during activation processes.

What are the potential research applications of studying cleaved MMP27 across different biological systems?

Cleaved MMP27 research has implications in multiple biological contexts:

This antibody provides a valuable tool for detecting the activated form of MMP27 specifically, allowing researchers to distinguish between inactive and active enzyme pools in these various contexts .

What strategies can be employed to extend this antibody's applications beyond Western Blot and ELISA?

While currently validated primarily for Western Blot and ELISA , researchers might explore these optimization strategies for additional applications:

• Immunohistochemistry (IHC) optimization:

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Try different fixation protocols (formalin, methanol, acetone)

  • Optimize antibody concentration (starting with 1:50-1:200 dilutions)

  • Test various detection systems (ABC, polymer-based)

• Immunofluorescence (IF) adaptation:

  • Begin with protocols successful for other polyclonal antibodies

  • Test various fixation and permeabilization combinations

  • Use signal amplification systems if needed

  • Include appropriate controls to confirm specificity

• Immunoprecipitation (IP):

  • Conduct preliminary experiments using various lysis buffers

  • Optimize antibody:protein ratios

  • Consider cross-linking the antibody to beads

  • Verify results with Western Blot analysis

• Flow cytometry potential:

  • Test permeabilization protocols to access intracellular epitopes

  • Optimize antibody concentration for reduced background

  • Use appropriate fluorophore-conjugated secondary antibodies

Always validate new applications thoroughly with proper controls, including blocking peptide experiments .

How can I troubleshoot inconsistent results when using Cleaved-MMP27 (Y99) Antibody across different experimental batches?

For improving reproducibility:

• Standardize sample preparation:

  • Use consistent lysis buffers and protein extraction protocols

  • Standardize protein quantification methods

  • Prepare fresh samples whenever possible or aliquot and store consistently

• Antibody handling:

  • Aliquot antibody upon receipt to avoid repeated freeze-thaw cycles

  • Maintain consistent antibody dilutions across experiments

  • Use the same diluent buffer for all experiments

• Protocol standardization:

  • Document detailed protocols including incubation times and temperatures

  • Use automated systems where possible to reduce human error

  • Maintain consistent reagent sources and lots when feasible

• Quality control measures:

  • Include positive and negative controls in every experiment

  • Run internal standards to normalize between experiments

  • Consider using loading controls for quantitative comparisons

• Environmental factors:

  • Control laboratory temperature and humidity

  • Minimize exposure to light for light-sensitive reagents

  • Use filtered water and high-quality reagents

What are the latest research findings involving MMP27 that could influence how this antibody is utilized?

Recent research developments suggest expanded applications for Cleaved-MMP27 (Y99) Antibody:

• MMP27 in cancer progression: Studies indicate MMP27 may play roles in tumor invasion and metastasis through extracellular matrix degradation, suggesting this antibody could be valuable in cancer research for tracking MMP27 activation states .

• Post-translational modifications: MMP27 undergoes N-glycosylation , which may affect antibody recognition. Researchers should consider how these modifications might impact detection when designing experiments.

• Subcellular localization insights: Unlike most MMPs which are secreted, MMP27 is retained in the endoplasmic reticulum due to its C-terminal extension . This unique localization suggests potential roles in intracellular protein processing, opening new research directions.

• Regulatory mechanisms: Understanding the specific activation triggers for MMP27 cleavage at Y99 could provide insights into disease mechanisms where inappropriate MMP activation occurs.

• Comparative studies across species: While currently validated for human and monkey samples , researchers are exploring cross-reactivity with other species, potentially expanding the antibody's utility in comparative biology and model organism research.