GZMM Antibody

What is Granzyme M (GZMM) and what is its role in the immune system?

Granzyme M (GZMM) is a serine protease primarily known for its role in immune-mediated apoptotic pathways. It functions by cleaving peptide substrates preferentially after methionine, leucine, and norleucine residues. Its physiological substrates include ezrin (EZR), alpha-tubulins, and the apoptosis inhibitor BIRC5/Survivin. GZMM promotes caspase activation and subsequent apoptosis of target cells, contributing to the cytotoxic function of natural killer cells and certain T lymphocytes . Unlike other granzymes, GZMM has been increasingly recognized for its involvement in inflammatory responses beyond direct cytotoxicity, suggesting a multifaceted role in immune regulation .

What types of GZMM antibodies are available for research purposes?

GZMM antibodies are predominantly available as polyclonal antibodies produced in rabbits, though some monoclonal options exist. These antibodies can be obtained in different preparations:

Most commercially available GZMM antibodies target the central region of the protein (aa 50-250) and are suitable for applications including Western blotting (1:250-1:500 dilution), immunohistochemistry (1:50-1:100), immunofluorescence (1:100), and flow cytometry (1:10-1:50) .

How is GZMM expression distributed across different cell types?

While traditionally associated with natural killer cells and cytotoxic T lymphocytes, research has revealed GZMM expression in unexpected cell populations. Recent studies have demonstrated that GZMM is expressed in various cancer cell lines independent of perforin, including human cancer cell lines (HT29, HepG2, PC-3, PC-3M) and murine carcinoma cell lines . This expression pattern suggests functions beyond the canonical cytotoxic role. Notably, the highly metastatic PC-3M prostate cancer cell line exhibited higher GZMM expression than its parent PC-3 line, indicating a potential association with metastatic potential . When investigating GZMM expression, researchers should consider both immune and non-immune cell sources, particularly in tumor microenvironments where its expression may have distinct biological significance.

What are the optimal protocols for detecting GZMM using immunohistochemistry?

For optimal GZMM detection in paraffin-embedded tissues, the following methodology is recommended:

• Sample preparation: Fix tissue samples in 10% neutral buffered formalin for 24-48 hours, followed by paraffin embedding using standard protocols.

• Sectioning and antigen retrieval: Prepare 4-5μm sections and perform heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95-98°C for 15-20 minutes.

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5% normal goat serum for 1 hour

  • Incubate with anti-GZMM antibody (1:50-1:100 dilution) overnight at 4°C

  • Incubate with appropriate secondary antibody for 1 hour at room temperature

• Signal development: Develop signal using DAB substrate and counterstain with hematoxylin.

For validation, human lung tissue has been successfully used to demonstrate GZMM expression patterns, with antibodies such as ab234746 working effectively at 1:100 dilution . Multiple independent fields should be analyzed, with appropriate positive and negative controls included to validate staining specificity.

How can I optimize Western blot conditions for GZMM detection?

GZMM detection by Western blot requires specific optimization due to its relatively low expression levels in some systems. The following protocol enhancements improve detection sensitivity:

• Sample preparation:

  • Add protease inhibitors immediately after cell lysis

  • Use RIPA buffer supplemented with 1% Triton X-100 for efficient extraction

  • Concentrate samples if necessary using TCA precipitation

• Gel electrophoresis and transfer:

  • Use 12-15% polyacrylamide gels for optimal resolution of the ~27.5 kDa GZMM protein

  • Transfer at lower voltage (80V) for extended time (90 minutes) in 20% methanol transfer buffer

• Antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary anti-GZMM antibody at 1:250-1:500 dilution overnight at 4°C

  • Use high-sensitivity detection systems (ECL-Plus or fluorescent secondary antibodies)

• Controls and validation:

  • Include positive controls (peripheral blood mononuclear cells)

  • Use GZMM knockdown or overexpression samples for specificity validation

For consistent results, avoid repeated freeze-thaw cycles of samples and optimize antibody concentrations for your specific cell type, as expression levels vary significantly between different cellular contexts .

What controls should be included when using GZMM antibodies for immunofluorescence?

When performing immunofluorescence with GZMM antibodies, a comprehensive set of controls should be included to ensure result reliability:

• Positive tissue/cell controls: Human peripheral blood mononuclear cells (PBMCs) serve as excellent positive controls due to their consistent GZMM expression. For cancer research, validated GZMM-expressing cell lines like HT29 or HepG2 can be used .

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Peptide competition assay using the immunizing peptide

  • Cells with confirmed GZMM knockdown (if available)

• Dual-labeling controls:

  • Single-labeled samples for each fluorophore to assess bleed-through

  • Co-staining with established NK or cytotoxic T cell markers (CD56, CD8) to confirm expected cellular localization

  • Co-staining with organelle markers to validate subcellular localization (typically cytoplasmic granular pattern)

• Technical validation:

  • Secondary antibody-only controls to assess non-specific binding

  • Autofluorescence controls, particularly important for tissues with high endogenous fluorescence

For optimal results with MCF7 cells, a 1:100 dilution of GZMM antibody with Alexa Fluor 488-conjugated secondary antibody has been demonstrated to provide specific cytoplasmic staining . Include DAPI nuclear counterstain and capture images using standardized exposure settings across all experimental conditions.

How can GZMM antibodies be used to investigate its role in tumor progression and chemoresistance?

GZMM antibodies provide valuable tools for examining the emerging role of GZMM in cancer biology. Recent research has revealed GZMM's unexpected contribution to chemoresistance, invasion, and metastasis. A comprehensive investigation approach includes:

• Expression analysis in chemoresistant models:

  • Use Western blotting and immunohistochemistry with GZMM antibodies to compare expression in parental versus chemoresistant cell lines

  • Quantify GZMM upregulation following chemotherapy treatment (e.g., 5-FU, doxorubicin, cisplatin)

  • Correlate GZMM expression with established chemoresistance markers

• Functional validation through genetic manipulation:

  • Create stable GZMM knockdown and overexpression models as demonstrated in murine tumor cell lines

  • Apply GZMM antibodies to confirm successful manipulation at the protein level

  • Assess proliferation, colony formation, and chemosensitivity in these models

• Invasion and metastasis studies:

  • Utilize immunofluorescence with GZMM antibodies to assess localization during invasion

  • Examine GZMM expression in primary tumors versus metastatic lesions using immunohistochemistry

  • Correlate GZMM expression with EMT markers in clinical samples

• Mechanistic investigations:

  • Use co-immunoprecipitation with GZMM antibodies to identify interaction partners

  • Perform chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify potential transcriptional targets

Research has demonstrated that GZMM overexpression promotes colony formation and heightens resistance to common chemotherapeutics, while knockdown enhances chemosensitivity . This approach has successfully revealed GZMM's role in promoting STAT3 activation and epithelial-mesenchymal transition in cancer cells.

What methodologies can distinguish between GZMM expressed by tumor cells versus immune infiltrates?

Distinguishing the source of GZMM expression in tumor microenvironments presents a significant challenge requiring sophisticated techniques:

• Multicolor immunofluorescence/immunohistochemistry:

  • Triple staining with antibodies against GZMM, tumor markers (e.g., cytokeratin), and immune cell markers (CD56 for NK cells)

  • Use spectrally distinct fluorophores and capture using multispectral imaging systems

  • Apply tissue cytometry for quantitative assessment of co-localization

• Laser capture microdissection combined with protein analysis:

  • Identify regions of interest using immunofluorescence

  • Microdissect tumor nests and immune infiltrates separately

  • Perform Western blotting with GZMM antibodies on isolated cellular populations

• Single-cell analysis approaches:

  • Disaggregate tumor tissue into single-cell suspensions

  • Perform flow cytometry using GZMM antibodies combined with lineage markers

  • Analyze by mass cytometry (CyTOF) for higher-dimensional protein profiling

• In situ hybridization combined with immunohistochemistry:

  • RNAscope for GZMM mRNA detection

  • Sequential immunohistochemistry for cell type-specific markers

  • Digital overlays to determine cellular source of expression

These techniques have revealed that GZMM expression occurs in both immune cells and tumor cells independently. Notably, in prostate cancer models, the highly metastatic PC-3M cell line exhibits higher GZMM expression than the less aggressive PC-3 line, suggesting a potential correlation with metastatic capability . When applying these methods, careful titration of antibodies and appropriate controls are essential to avoid false positive signals.

How can GZMM antibodies be applied to investigate its non-apoptotic functions in inflammation and tissue remodeling?

GZMM exhibits functions beyond its canonical role in cytotoxicity, with emerging evidence suggesting contributions to inflammation and tissue remodeling. To investigate these non-canonical functions:

• Cytokine regulation studies:

  • Use GZMM antibodies to immunoprecipitate the protein from conditioned media

  • Perform parallel ELISA assays to correlate GZMM levels with inflammatory cytokine profiles

  • Analyze cytokine production in GZMM-manipulated cells using multiplexed bead-based assays

• Extracellular matrix degradation assessment:

  • Conduct zymography assays combining GZMM antibodies with substrate gels

  • Investigate co-localization of GZMM with matrix components using dual immunofluorescence

  • Quantify matrix degradation patterns in the presence of GZMM inhibitors

• Inflammation models:

  • Apply immunohistochemistry with GZMM antibodies to tissue sections from inflammatory disease models

  • Correlate GZMM expression with inflammatory markers and tissue damage scores

  • Examine GZMM localization relative to infiltrating immune cells and damaged tissue areas

• Proteomic approaches to identify substrates:

  • Use GZMM antibodies for activity-based protein profiling

  • Perform targeted proteomics to identify cleaved substrates in GZMM-expressing systems

  • Validate identified substrates using in vitro cleavage assays with recombinant GZMM

Research has shown that GZMM-expressing tumor cells secrete higher levels of inflammatory cytokines, potentially contributing to a pro-tumorigenic microenvironment. Additionally, GZMM's proteolytic activity appears to facilitate tumor invasion through extracellular matrix degradation, as demonstrated in transwell invasion assays where GZMM knockdown significantly reduced invasive capacity . These methodologies help distinguish GZMM's direct enzymatic functions from its signaling roles in non-apoptotic contexts.

How can I address non-specific binding when using GZMM antibodies?

Non-specific binding is a common challenge when working with GZMM antibodies, particularly in tissues with high endogenous protease activity. Systematic optimization can significantly improve specificity:

• Sample preparation optimization:

  • Fresh samples yield better results than archived material

  • Optimize fixation time (12-24 hours for tissues) to prevent antigen masking

  • For frozen sections, post-fix in acetone for 10 minutes to preserve antigenic epitopes

• Blocking optimization:

  • Test multiple blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to 2 hours at room temperature

  • Use dual blocking approach: protein block followed by Fc receptor block

  • Add 0.1-0.3% Triton X-100 during blocking for better antibody penetration

• Antibody optimization:

  • Titrate antibody concentrations (recommended range: 1:50-1:500)

  • Extend primary antibody incubation to overnight at 4°C with gentle agitation

  • Use antibody diluents containing stabilizing proteins and mild detergents

  • Pre-adsorb antibody with tissue powder from species of sample origin

• Washing optimization:

  • Extend wash times (5 washes, 5 minutes each)

  • Use PBS-T with 0.1% Tween-20 for more stringent washing

  • Include a high-salt wash (PBS with 500mM NaCl) as the second wash

When troubleshooting, compare staining patterns across different antibody clones or suppliers, as epitope accessibility may vary. If problems persist, peptide competition assays provide definitive evidence of specificity. For Western blotting, the expected molecular weight of GZMM is approximately 27.5 kDa, with potential glycosylated forms appearing at slightly higher molecular weights .

How should I interpret discrepancies in GZMM expression patterns between different detection methods?

Discrepancies in GZMM detection across different methodologies are common and require careful interpretation:

• Method-specific considerations:

  Method	Sensitivity	Specificity	Common Discrepancies
  RT-PCR	High	Moderate	Detects mRNA but not protein; may not reflect active protein levels
  Western blot	Moderate	High	May miss low abundance expression; denaturation can affect epitope recognition
  IHC/IF	Moderate	Variable	Fixation-dependent; may show false positives due to endogenous peroxidases
  Flow cytometry	High	High	Requires permeabilization; may detect intracellular pools not represented in other methods

• Biological explanations for discrepancies:

  • Post-transcriptional regulation may explain mRNA-protein discordance

  • Subcellular localization differences may affect antibody accessibility

  • Protein degradation during sample processing can reduce detection

  • Alternative splicing or post-translational modifications may affect epitope availability

• Validation strategies:

  • Confirm findings with at least two independent detection methods

  • Use genetic manipulation (knockdown/overexpression) to validate antibody specificity

  • Apply antibodies targeting different epitopes to confirm expression patterns

  • Include appropriate positive controls (PBMCs) alongside experimental samples

Research has shown that some cancer cell lines (e.g., PC-3) may express GZMM mRNA but show minimal protein expression by flow cytometry, while others (PC-3M, HepG2, HT29) demonstrate both mRNA and protein expression . When faced with discrepancies, consider that the subcellular localization of GZMM may affect its detection, as it can exist in both soluble cytoplasmic forms and membrane-associated compartments.

What are the key considerations when quantifying GZMM expression for comparative studies?

Accurate quantification of GZMM expression for comparative studies requires rigorous methodological considerations:

• Standardization of sample collection and processing:

  • Establish consistent timing between sample collection and processing

  • Standardize fixation protocols (duration, temperature, fixative composition)

  • Process all samples within a comparative study simultaneously

• Quantification approaches for different techniques:

  • Western blot: Use densitometry with normalization to multiple housekeeping proteins

  • qRT-PCR: Apply the ΔΔCt method with validation of reference gene stability

  • IHC/IF: Implement digital image analysis with standardized algorithms

  • Flow cytometry: Report median fluorescence intensity and percent positive cells

• Controls for normalization and calibration:

  • Include calibration standards across multiple plates/gels/sections

  • Prepare a reference sample to run on each experimental batch

  • For IHC, use tissue microarrays to minimize staining variability

• Statistical considerations:

  • Determine appropriate sample sizes through power analysis

  • Account for technical and biological replicates in statistical models

  • Apply appropriate transformations for non-normally distributed data

  • Use paired statistical tests when comparing the same samples across conditions

When quantifying GZMM in tumor samples, it's critical to account for immune infiltration, as GZMM expressed by tumor-infiltrating lymphocytes can confound analysis of tumor cell expression. Research exploring chemoresistance demonstrated that 5-FU treatment induced a 2-3 fold increase in GZMM expression in CT26 and 4T1 cells, highlighting the importance of standardized treatment conditions when examining drug-induced changes in expression . Additionally, establish clear criteria for defining "high" versus "low" expression based on validated thresholds with clinical or functional relevance.

How can GZMM antibodies be utilized to study its role in the tumor microenvironment and immune evasion?

GZMM's emerging role in cancer biology extends to potential immune regulatory functions within the tumor microenvironment. Advanced applications of GZMM antibodies include:

• Spatial proteomics approaches:

  • Multiplex immunofluorescence combining GZMM with immune checkpoint markers (PD-1, PD-L1)

  • Digital spatial profiling to map GZMM expression relative to tumor and immune cell populations

  • Correlation of GZMM expression patterns with immune infiltration characteristics

• Functional immunological assays:

  • Ex vivo tumor slice cultures with GZMM-neutralizing antibodies to assess immune cell function

  • T-cell killing assays comparing GZMM-high versus GZMM-low tumor targets

  • Analysis of antigen presentation efficiency in GZMM-manipulated cancer cells

• In vivo immune modulation studies:

  • Therapeutic administration of GZMM-neutralizing antibodies in tumor models

  • Assessment of tumor-infiltrating lymphocyte profiles following GZMM modulation

  • Combination therapies targeting GZMM alongside established immunotherapies

• Clinical correlative studies:

  • Immunohistochemistry analysis of GZMM in responders versus non-responders to immunotherapy

  • Prospective biomarker studies using GZMM antibodies in immunotherapy trials

  • Liquid biopsy approaches to detect circulating GZMM as a biomarker

Early evidence suggests that tumor-derived GZMM may influence cytokine profiles that potentially modulate immune cell function in the tumor microenvironment. Experiments have shown that GZMM overexpression in tumor cells leads to increased production of inflammatory mediators , which could contribute to both pro-tumor inflammation and immune suppression. These approaches may uncover novel therapeutic opportunities targeting the non-canonical functions of GZMM in cancer.

What are the considerations for developing GZMM-specific inhibitors for therapeutic applications?

The development of GZMM-specific inhibitors represents an emerging area with potential therapeutic applications in cancer and inflammatory conditions. Key considerations include:

• Target validation strategies using antibodies:

  • Neutralizing antibodies against GZMM to validate therapeutic potential

  • Immunoprecipitation with GZMM antibodies followed by activity assays to assess inhibitor specificity

  • Immunofluorescence to track cellular localization changes following inhibition

• Inhibitor screening approaches:

  • Development of fluorogenic peptide substrates based on GZMM's preference for Met/Leu at P1

  • High-throughput screening with recombinant GZMM and validated substrates

  • Secondary validation in cell-based assays with GZMM antibody detection

• Selectivity assessment:

  • Cross-reactivity testing against related serine proteases (granzymes A, B, K)

  • Structural considerations targeting the unique S1 pocket of GZMM

  • Cellular protease profiling using activity-based protein probes

• Delivery and efficacy monitoring:

  • Targeted delivery systems to GZMM-expressing tumors

  • Pharmacodynamic monitoring using GZMM antibodies in tissue biopsies

  • Correlation of GZMM inhibition with reversal of chemoresistance phenotypes

Building on research demonstrating GZMM's role in promoting chemoresistance , inhibitor development holds promise for sensitizing resistant tumors to conventional therapies. When designing GZMM inhibitors, researchers must consider the unique substrate preferences and structural features that distinguish GZMM from other granzymes, particularly its preference for cleaving after methionine residues. Antibody-based approaches provide critical tools for validating target engagement and biological responses to candidate inhibitors.