MMP28 Antibody, HRP conjugated

What is MMP28 and what are its primary functions in human tissues?

MMP28 (Epilysin) is a secreted enzyme that degrades casein and plays important roles in tissue homeostasis and wound repair. It belongs to the matrix metalloproteinase family, which is involved in the breakdown of extracellular matrix for normal physiological processes such as embryonic development, reproduction, and tissue remodeling, as well as disease processes including asthma and cancer metastasis . MMP28 is highly expressed in skin basal cells, keratinocytes in the upper basement membrane, developing spermatogonium in testis, and lung tissue . At the protein level, MMP28 shows relatively high expression in the lung, heart, rectum, small intestine, and brain .

What are the key specifications of MMP28 Antibody, HRP conjugated?

MMP28 Antibody, HRP conjugated is typically available as a polyclonal antibody with the following specifications:

This information is critical for researchers to evaluate if the antibody is suitable for their experimental design and conditions .

What applications is MMP28 Antibody, HRP conjugated best suited for?

• Western blot analysis - Particularly useful for detecting MMP28 in cell lysates and tissue samples with predicted band sizes around 48-59 kDa

• Immunocytochemistry - Can be used to visualize MMP28 cellular localization

• Flow cytometry - When proper cell fixation and permeabilization protocols are followed

For optimal results, researchers should determine the ideal dilutions/concentrations for their specific experimental conditions . The dilution range typically varies between 1:200-1:1000 for Western blotting applications .

How should MMP28 Antibody, HRP conjugated be stored and handled for optimal performance?

For optimal performance and longevity of the MMP28 Antibody, HRP conjugated:

• Store the antibody at -20°C in aliquots to minimize freeze-thaw cycles

• Avoid exposure to light as HRP is light-sensitive

• Avoid repeated freeze/thaw cycles which can lead to denaturation and decreased activity

• Store in manufacturer-recommended buffer (typically 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol)

• When working with the antibody, keep it on ice and return to -20°C as soon as possible

• Prior to use, centrifuge the antibody vial briefly to collect contents at the bottom of the tube

Following these guidelines will help maintain antibody integrity and ensure consistent experimental results .

How does MMP28 expression vary across different tissue types and disease states?

MMP28 expression shows distinctive patterns across various tissues and pathological conditions:

This varied expression pattern across different tissues and disease states makes MMP28 an important target for research into tissue homeostasis, wound healing, immune function, and cancer progression .

What are the recommended protocols for using MMP28 Antibody, HRP conjugated in Western blot analysis?

For optimal Western blot results with MMP28 Antibody, HRP conjugated:

Protocol:

• Sample preparation: Prepare cell or tissue lysates using standard methods. Use 50μg of protein per lane for optimal detection.

• SDS-PAGE: Perform electrophoresis on a 5-20% SDS-PAGE gel at 70V (stacking gel) / 90V (resolving gel) for 2-3 hours.

• Transfer: Transfer proteins to a nitrocellulose membrane at 150mA for 50-90 minutes.

• Blocking: Block the membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature.

• Primary antibody incubation: Dilute MMP28 antibody to 0.5-1.0 μg/mL in blocking buffer and incubate overnight at 4°C.

• Washing: Wash membrane with TBS-0.1% Tween 3 times, 5 minutes each.

• Development: Develop signal using an enhanced chemiluminescent detection (ECL) kit.

Expected Results:

• MMP28 typically appears as a band at approximately 48-59 kDa, with the pro-form at around 58 kDa and the active (processed) form at around 50 kDa.

• The C-terminal domain may also be detected at approximately 33 kDa.

Sample Western blot data:
Human placenta tissue lysates and A549 whole cell lysates typically show strong MMP28 expression . The pro form is often predominant in cell lysates, whereas in conditioned medium and the ECM, the active form may be predominant .

How can researchers validate the specificity of MMP28 Antibody, HRP conjugated?

To validate MMP28 Antibody specificity:

• Positive and negative controls: Use tissues or cell lines known to express MMP28 (A549 cells, human placenta, testis, skin) as positive controls. Use tissues known to express low levels of MMP28 or MMP28 knockout samples as negative controls.

• Blocking peptide: Perform a parallel experiment where the antibody is pre-incubated with the blocking peptide (immunogen) to confirm specific binding.

• siRNA knockdown: Compare antibody reactivity in cells with normal MMP28 expression versus cells with MMP28 knockdown using siRNA.

• Western blot analysis: Verify that the detected band corresponds to the expected molecular weight (approximately 48-59 kDa for MMP28) and compare with recombinant MMP28 protein as a standard.

• Cross-reactivity testing: Test the antibody against other MMPs to ensure specificity.

These validation steps are critical for ensuring the reliability and reproducibility of experimental results, especially in research focused on MMP28's functional roles in different physiological and pathological conditions .

What is the relationship between MMP28 and TGF-β signaling pathways?

MMP28 has been shown to interact significantly with TGF-β signaling pathways:

• Regulatory relationship: In several studies, overexpression of MMP28 induced TGF-β protein expression, while downregulation of MMP28 suppressed TGF-β protein expression in glioma cells. This suggests a direct regulatory relationship between MMP28 and TGF-β .

• Epithelial-mesenchymal transition (EMT): In lung adenocarcinoma, high MMP28 expression can activate the TGF-β pathway, thereby inducing epithelial-mesenchymal transition of epithelial cells .

• Cell growth and apoptosis: The MMP28/TGF-β axis affects cell growth and apoptosis in glioma cells. Upregulation of MMP28 induced cell growth and reduced apoptosis by activating TGF-β, while downregulation of MMP28 reduced cell growth and increased apoptosis via suppression of TGF-β .

• Intervention studies: TGF-β inhibitors have been shown to attenuate the effects of MMP28 in glioma cells, further confirming the functional relationship between these molecules .

• Expression correlation: In certain cancer types, there is a positive correlation between MMP28 and TGF-β expression levels, suggesting coordinated regulation or functional interaction .

This relationship highlights the potential importance of MMP28 as a therapeutic target in diseases where TGF-β signaling plays a critical role .

What are the post-translational modifications of MMP28 and how do they affect its function?

MMP28 undergoes several important post-translational modifications that significantly impact its function:

• Proprotein convertase processing: MMP28 contains a furin activation sequence within its propeptide domain, suggesting intracellular activation. Studies have confirmed that MMP28 processing is mediated by furin-like proprotein convertases, converting the pro-form (approximately 58 kDa) to the active form (approximately 50 kDa) .

• Differential compartmentalization: The distribution of MMP28 forms varies between cellular compartments. The pro-form is predominantly found in cell lysates, while the active (processed) form is mainly associated with the extracellular matrix (ECM) .

• C-terminal domain processing: The C-terminal domain of MMP28 (approximately 33 kDa) can be detected separately and may play a role in the protein's localization and function .

• ECM association: Active MMP28 preferentially associates with the ECM in a C-terminal independent manner. This ECM association can be disrupted by treatment with heparin, suggesting the interaction is mediated by heparan sulfate proteoglycans .

• Cell surface binding: MMP28 can also localize to the cell surface transiently, in a C-terminal dependent manner. This localization may be important for its function in specific contexts .

Understanding these post-translational modifications is crucial for researchers using MMP28 antibodies, as different antibodies may recognize different forms or epitopes of the protein, potentially leading to variable results across experimental systems .

How can researchers distinguish between latent and active forms of MMP28 using antibody-based methods?

Distinguishing between latent (pro) and active forms of MMP28 requires specific methodological approaches:

• Western blot analysis with size discrimination:

  • The pro-form of MMP28 appears at approximately 58 kDa

  • The active form appears at approximately 50 kDa

  • Use of gradient gels (5-20% SDS-PAGE) can improve resolution between these forms

• Domain-specific antibodies:

  • Antibodies targeting the pro-domain will only detect the latent form

  • Antibodies targeting the catalytic domain will detect both forms

  • C-terminal domain antibodies can detect both forms plus the cleaved C-terminal domain (~33 kDa)

• Activity-based probes:

  • Use of activity-based probes that bind only to the active site of functional MMPs

  • These can be coupled with immunoprecipitation using MMP28-specific antibodies

• Zymography with immunoblotting:

  • Perform casein zymography (as casein is a known substrate)

  • Follow with immunoblotting using MMP28 antibodies to confirm identity

• Cell compartment analysis:

  • Separate analysis of cell lysates (predominantly pro-form)

  • Analysis of ECM fractions (predominantly active form)

  • Analysis of conditioned media (mixture of forms)

This multi-approach strategy provides researchers with robust methods to differentiate between the MMP28 forms, which is critical for functional studies where the activation state of MMP28 is a key variable .

What are the emerging roles of MMP28 in cancer progression and how can MMP28 antibodies be used to study these roles?

MMP28 exhibits complex roles in cancer progression that vary by cancer type:

Research applications for MMP28 antibodies in cancer research:

• Tissue microarray analysis: To evaluate MMP28 expression across large patient cohorts and correlate with clinical outcomes

• Cellular localization studies: Using immunofluorescence to examine changes in MMP28 localization during cancer progression

• Functional studies: Using antibodies to neutralize MMP28 function in in vitro and in vivo models

• Therapeutic targeting: Development of antibody-based therapies targeting MMP28 in cancers where it promotes progression

• Biomarker development: Using MMP28 antibodies in diagnostic assays to assess cancer progression risk

How does MMP28 interact with the extracellular matrix and what methodologies can detect these interactions?

MMP28 exhibits specific interaction patterns with the extracellular matrix (ECM):

• Preferential ECM association:

  • Active (processed) MMP28 preferentially associates with the ECM compared to the pro-form

  • This association occurs in a C-terminal independent manner

  • The ECM association can be disrupted by treatment with heparin, suggesting binding occurs via heparan sulfate proteoglycans

• Effect on cell-ECM interactions:

  • Overexpression of MMP28 in chondrosarcoma cells leads to altered cell morphology with increased organization of actin

  • Adhesion to type II collagen and fibronectin is increased

  • Migration across type II collagen is decreased

  • These changes suggest MMP28 modulates cell-ECM interactions in ways beyond simple matrix degradation

• Cell surface localization:

  • MMP28 can localize to the cell surface transiently

  • This localization is C-terminal dependent

  • Heparin treatment prevents cell surface binding, suggesting binding via heparan sulfate proteoglycans

Methodologies to study MMP28-ECM interactions:

• Fractionation studies:

  • Separate analysis of ECM, cell lysate, and conditioned media fractions

  • Western blot analysis of each fraction to determine MMP28 distribution

  • Compare wild-type MMP28 with domain deletion mutants to identify regions responsible for specific interactions

• Competition assays:

  • Use of heparin or other glycosaminoglycans to compete with ECM binding

  • Dose-response studies to determine binding affinities

  • Analysis of conditioned media after treatment to quantify MMP28 release from ECM

• Immunofluorescence and confocal microscopy:

  • Co-localization studies with ECM components

  • Live-cell imaging to track MMP28 trafficking and ECM deposition

  • FRAP (Fluorescence Recovery After Photobleaching) to assess dynamics of MMP28-ECM interactions

• Surface plasmon resonance (SPR):

  • Direct measurement of binding kinetics between purified MMP28 and ECM components

  • Comparison of different MMP28 forms (pro vs. active)

• Proximity ligation assay (PLA):

  • In situ detection of MMP28 proximity to specific ECM components

  • Useful for detecting transient interactions in cellular contexts

These methodologies provide researchers with tools to understand the complex and dynamic interactions between MMP28 and the ECM, which may be critical to its biological functions beyond simple enzymatic activity .

What are the recommended protocols for immunohistochemistry using MMP28 antibodies?

For optimal immunohistochemistry (IHC) results with MMP28 antibodies:

Protocol:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-6 μm thickness

  • Mount sections on positively charged slides

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) or TE buffer (pH 9.0)

  • For MMP28, studies suggest TE buffer (pH 9.0) may yield better results

• Endogenous peroxidase blocking:

  • Incubate sections in 3% hydrogen peroxide for 10 minutes

  • Wash in PBS

• Blocking:

  • Block with 10% normal goat serum for 30 minutes at room temperature

• Primary antibody incubation:

  • Dilute MMP28 antibody 1:20-1:200 in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

• Detection system:

  • For HRP-conjugated antibodies, proceed directly to chromogen development

  • For unconjugated antibodies, use appropriate secondary antibody and detection system

• Chromogen development:

  • Develop with DAB (3,3'-diaminobenzidine) substrate

  • Monitor under microscope for optimal development (typically 5-10 minutes)

• Counterstaining and mounting:

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount with permanent mounting medium

Validation tissues:
Human kidney, ovary, skin cancer, and testis tissues have been validated for MMP28 IHC and show positive staining .

Scoring system:
For semi-quantitative analysis of MMP28 expression in tissues, the following scoring system has been validated:

• 0: No staining

• 1: Faint staining or moderate/strong staining in <25% of cells

• 2: Moderate staining or strong staining in 25-50% cells

• 3: Strong staining in >50% cells

This scoring system has been used successfully to correlate MMP28 expression with clinical outcomes in cancer studies .

How can researchers troubleshoot common problems when using MMP28 Antibody, HRP conjugated?

When working with MMP28 Antibody, HRP conjugated, researchers may encounter various issues. Here are troubleshooting approaches for common problems:

1. Weak or no signal in Western blot:

• Problem: Insufficient protein or antibody concentration

• Solution: Increase protein loading (50μg recommended) or antibody concentration

• Problem: Inefficient transfer

• Solution: Optimize transfer conditions for high molecular weight proteins

• Problem: HRP activity loss

• Solution: Avoid repeated freeze/thaw cycles and exposure to light

2. High background in immunoassays:

• Problem: Insufficient blocking

• Solution: Increase blocking time or concentration (5% milk/BSA)

• Problem: Too high antibody concentration

• Solution: Titrate antibody to determine optimal concentration

• Problem: Non-specific binding

• Solution: Pre-absorb antibody with control protein or use more stringent washing

3. Inconsistent detection of MMP28 forms:

• Problem: Different forms (pro vs. active) in different cell compartments

• Solution: Analyze cell lysates, conditioned media, and ECM fractions separately

• Problem: Processing during sample preparation

• Solution: Use protease inhibitors and process samples quickly at cold temperatures

4. Cross-reactivity issues:

• Problem: Antibody detecting related MMPs

• Solution: Validate specificity using recombinant proteins and consider using monoclonal antibodies

• Problem: Non-specific bands

• Solution: Optimize blocking conditions and antibody dilution

5. Immunohistochemistry issues:

• Problem: Poor antigen retrieval

• Solution: Compare different antigen retrieval methods (citrate vs. EDTA buffer)

• Problem: Tissue-specific fixation effects

• Solution: Optimize fixation time for different tissues

6. Variable results across experiments:

• Problem: Antibody degradation

• Solution: Store in small aliquots at -20°C and avoid repeated freeze/thaw

• Problem: Sample variation

• Solution: Include consistent positive controls in all experiments

• Problem: Protocol inconsistency

• Solution: Standardize protocols and limit variables between experiments

7. HRP-specific issues:

• Problem: Rapid signal decay

• Solution: Optimize development time and use fresh ECL substrate

• Problem: HRP inactivation

• Solution: Avoid sodium azide in buffers used with HRP-conjugated antibodies

Following these troubleshooting approaches can help researchers obtain consistent and reliable results when working with MMP28 Antibody, HRP conjugated .

How does MMP28 expression influence gene expression of other MMPs and TIMPs, and how can this be studied?

MMP28 expression has been shown to influence the expression of other matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Understanding these regulatory networks is crucial for comprehensive studies of extracellular matrix remodeling.

Effects of MMP28 on other MMPs and TIMPs:

• MMP2 regulation:

  • Overexpression of catalytically active MMP28 significantly increases both the expression and activity of MMP2

  • This effect is not observed with catalytically inactive EA mutant forms of MMP28, suggesting an enzyme activity-dependent mechanism

• MMP19 upregulation:

  • All forms of MMP28 tested (wild-type, EA mutant, and pro-cat) increase expression of MMP19 mRNA

  • This suggests that MMP28 regulates MMP19 through mechanisms independent of its catalytic activity

• TIMP3 upregulation:

  • MMP28 expression increases TIMP3 mRNA levels

  • This effect is observed with all forms of MMP28, including catalytically inactive mutants

  • The concurrent upregulation of both MMPs and TIMPs suggests MMP28 may play a complex role in maintaining ECM homeostasis

Methodologies to study these interactions:

• Quantitative RT-PCR analysis:

  • Compare expression levels of various MMPs and TIMPs in cells stably transfected with MMP28 versus control cells

  • Compare effects of wild-type MMP28 with catalytically inactive mutants to distinguish enzymatic from non-enzymatic effects

  • Example primer design for MMP28:
    Forward: 5'-CTCATCCTCTTCAAGGGTG-3' (nt 1,366 to 1,383)
    Reverse: 5'-GGAAGAAGATGATGGAGCCA-3' (nt 1,606 to 1,623)

• Zymography:

  • Assess MMP activity (particularly MMP2) in conditioned media from MMP28-expressing versus control cells

  • Gelatinolytic zymography for MMP2/9

  • Caseinolytic zymography for other MMPs

• Promoter-reporter assays:

  • Construct reporter plasmids containing promoter regions of MMP2, MMP19, and TIMP3

  • Co-transfect with MMP28 expression constructs to determine direct transcriptional effects

• Chromatin immunoprecipitation (ChIP):

  • Identify whether MMP28 influences transcription factor binding to promoters of other MMPs and TIMPs

• Pathway inhibition studies:

  • Use inhibitors of various signaling pathways to determine the mechanisms through which MMP28 regulates other genes

  • Compare effects of TGF-β pathway inhibitors, as MMP28 has been linked to TGF-β signaling

• Proteomics analysis:

  • Compare the secretome of MMP28-expressing versus control cells to identify broader effects on extracellular protein composition

These approaches provide researchers with tools to unravel the complex regulatory networks in which MMP28 participates, which is essential for understanding its role in both physiological and pathological processes .

What are the latest developments in using MMP28 as a biomarker or therapeutic target in various diseases?

Recent research has highlighted MMP28's potential as both a biomarker and therapeutic target across several disease contexts:

1. Cancer biomarker applications:

2. Therapeutic targeting approaches:

• TGF-β pathway modulation:
  The established relationship between MMP28 and TGF-β signaling suggests targeting this axis could be therapeutically beneficial:

  • In glioma, where MMP28 activates TGF-β, combination therapy targeting both molecules might enhance treatment efficacy

  • In lung adenocarcinoma, where MMP28 promotes epithelial-mesenchymal transition via TGF-β, blocking this interaction could inhibit metastasis

• Cell invasion and metastasis inhibition:
  In gastric cancer models, inhibiting MMP28 reduces invasion and metastasis, suggesting MMP28 inhibitors could serve as anti-metastatic agents .

• Immune system modulation:
  MMP28's expression in T lymphocytes suggests potential roles in immune responses, which could be leveraged in immunotherapy approaches .

3. Emerging research directions:

• Targeted antibody therapies:
  Development of function-blocking antibodies against MMP28, similar to those used in myelination studies where MMP28 function-blocking antibodies enhanced myelination .

• Small molecule inhibitors:
  Design of selective MMP28 inhibitors that avoid the pitfalls of earlier broad-spectrum MMP inhibitors.

• Diagnostic imaging:
  Use of labeled MMP28 antibodies for detection of MMP28-expressing tumors or tissues.

• Tissue regeneration applications:
  Based on MMP28's role in wound healing and tissue homeostasis, modulating its activity could enhance tissue repair in specific contexts.

The dual role of MMP28 across different disease contexts highlights the importance of tissue-specific and disease-specific approaches when considering it as either a biomarker or therapeutic target. Future research should focus on understanding the molecular mechanisms underlying these context-dependent functions to develop more effective diagnostic and therapeutic strategies .

What are the key considerations for researchers when selecting and using MMP28 Antibody, HRP conjugated in their experiments?

When selecting and using MMP28 Antibody, HRP conjugated, researchers should consider:

• Experimental application compatibility: While HRP-conjugated antibodies are ideal for ELISA, they may also be used for Western blot, immunohistochemistry, and other applications. Researchers should verify that the specific antibody has been validated for their intended application .

• Form recognition specificity: Different antibodies may preferentially recognize the pro-form (~58 kDa), active form (~50 kDa), or C-terminal domain (~33 kDa) of MMP28. Understanding which form(s) your antibody detects is critical for accurate interpretation of results .

• Epitope location: The epitope location will influence what forms of MMP28 are detected. Antibodies targeting the propeptide will only detect the latent form, while those targeting the catalytic or C-terminal domains may detect multiple forms .

• Species cross-reactivity: Verify the antibody's reactivity across species if working with non-human models. While many MMP28 antibodies react with human samples, some also cross-react with mouse and rat samples .

• Protocol optimization: Optimal dilutions/concentrations should be determined empirically for each specific application and sample type. Manufacturer recommendations provide starting points but may need adjustment .

• Cellular compartment analysis: MMP28 distributes differently between cell lysates, conditioned media, and ECM fractions. Comprehensive analysis should include all relevant fractions to avoid missing important aspects of MMP28 biology .

• Controls: Include appropriate positive controls (A549 cells, human placenta, colon, or testis tissues) and negative controls (ideally MMP28 knockdown or knockout samples) .