GZMB Antibody

What is Granzyme B and why is it significant in immunological research?

Granzyme B (GZMB) is a potent cytotoxic serine protease predominantly produced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It plays a critical role in cell-mediated immune responses by inducing apoptosis in target cells such as tumor cells and infected cells. GZMB is significant in immunological research because it serves as a key effector molecule in cell-mediated cytotoxicity and has emerging non-canonical functions beyond direct cytotoxicity . The protein's official name is granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1), with a calculated molecular weight of approximately 28 kDa (247 amino acids), though it may be observed at around 33 kDa in experimental conditions .

What are the common applications of GZMB antibodies in research?

GZMB antibodies are utilized across multiple experimental platforms including:

These applications enable researchers to investigate GZMB expression, localization, and function in various experimental contexts .

What cell types express GZMB and how does this influence antibody selection?

While primarily expressed by CTLs and NK cells, recent evidence indicates GZMB expression in numerous other cell types:

• CD34+ hematopoietic progenitor cells

• Keratinocytes

• Basophils

• Mast cells

• Plasmacytoid dendritic cells

• B cells

• Smooth muscle cells

• Myeloid-derived suppressor cells (MDSCs)

When selecting antibodies, researchers should consider the cellular context, as GZMB expression varies significantly between cell types. For studying non-classical GZMB-producing cells, antibodies with high sensitivity and specificity are essential, potentially requiring optimization of staining protocols for each cell type .

How should I select the appropriate GZMB antibody for my specific application?

Selection criteria should be based on:

• Target application: Different antibodies perform optimally in specific applications. For instance, some antibodies work well in Western blot but poorly in IHC.

• Species reactivity: Confirm reactivity with your experimental species. Many GZMB antibodies are human-specific, while others cross-react with mouse, rat, or non-human primate samples .

• Clonality:

  • Monoclonal antibodies offer high specificity and consistency between lots but may have limited epitope recognition

  • Polyclonal antibodies provide broader epitope recognition but potential batch-to-batch variation

• Conjugation needs: Select unconjugated antibodies for flexibility in detection methods or pre-conjugated antibodies (fluorescent dyes, enzymes) for direct detection .

• Validated performance: Review validation data from manufacturers and literature citations demonstrating the antibody's performance in your application of interest .

What are the recommended protocols for GZMB detection in tissue sections?

For optimal GZMB detection in FFPE tissues:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section at 4-5 μm thickness

• Antigen retrieval:

  • High pH (pH 9.0) EDTA buffer typically yields superior results compared to citrate buffer

  • Heat-induced epitope retrieval using pressure cooker or microwave methods (20 minutes)

• Blocking and antibody incubation:

  • Block with appropriate serum (5-10%) for 30-60 minutes

  • Incubate with primary antibody at optimal dilution (typically 1:500-1:2000) overnight at 4°C

  • Use appropriate detection system (e.g., polymer-based or biotinylated secondary antibodies)

• Counterstaining and controls:

  • Include positive controls (tonsil or lymph node tissues) where GZMB+ cells are abundant

  • Implement negative controls (isotype or no primary antibody)

  • Counterstain with hematoxylin to visualize tissue architecture .

How do I optimize intracellular staining for GZMB in flow cytometry?

Effective intracellular GZMB staining requires careful optimization:

• Cell preparation:

  • Use freshly isolated cells or properly cryopreserved samples

  • Include viability dye to exclude dead cells

• Stimulation considerations:

  • For maximal GZMB detection, consider stimulation with PMA/ionomycin, high-dose IL-2, or IL-15

  • Include protein transport inhibitors (e.g., brefeldin A) to prevent secretion

• Fixation and permeabilization:

  • Fix cells with paraformaldehyde (1-4%) for 10-20 minutes

  • Permeabilize with saponin-based buffers for optimal intracellular access

  • For GZMB in granules, stronger permeabilization may be required (0.1% Triton X-100)

• Antibody staining:

  • Titrate antibody concentration (typically 0.25 μg per 10^6 cells)

  • Include appropriate fluorescent minus one (FMO) controls

  • Maintain permeabilization reagent in all wash steps

• Analysis considerations:

  • Implement proper compensation when using multiple fluorophores

  • Consider co-staining with perforin or other granule markers .

How can GZMB antibodies be used to study the non-canonical functions of Granzyme B?

Beyond cytotoxicity, GZMB plays roles in extracellular matrix remodeling, inflammation, and tissue development. To investigate these functions:

• Dual immunofluorescence staining:

  • Combine GZMB antibodies with markers for extracellular matrix proteins (collagens, fibronectin)

  • Use confocal microscopy to visualize co-localization of GZMB with potential substrates

• Tissue degradation assays:

  • Apply purified GZMB or GZMB-expressing cells to matrix-coated surfaces

  • Detect matrix degradation using complementary antibodies against cleaved matrix proteins

  • Implement GZMB inhibitors as controls to confirm specificity

• In vitro cleavage assays:

  • Incubate recombinant GZMB with potential substrate proteins

  • Use Western blot with appropriate antibodies to detect cleavage products

  • Confirm through mass spectrometry analysis of cleavage sites

• In vivo models:

  • Utilize tissue samples from GZMB knockout models to examine matrix integrity

  • Compare with samples from GZMB-overexpressing conditions

  • Implement immunohistochemistry to visualize tissue architecture and cell populations .

What approaches can differentiate between active and inactive forms of GZMB using antibodies?

Distinguishing active from inactive GZMB is crucial for functional studies:

• Activity-based probes:

  • Combine antibodies with activity-based serine protease probes

  • Use flow cytometry or microscopy to correlate GZMB protein levels with enzymatic activity

• Conformation-specific antibodies:

  • Some antibodies preferentially recognize the active conformation of GZMB

  • Compare staining patterns between antibodies recognizing different epitopes

• Granzyme activity assays:

  • Utilize fluorogenic substrates specific for GZMB activity in conjunction with antibody staining

  • Correlate substrate cleavage with antibody-detected protein levels

• Endogenous inhibitor detection:

  • Co-stain for GZMB and Serpin9 (its endogenous inhibitor)

  • Analyze the ratio between the protease and its inhibitor to infer potential activity levels .

How can GZMB antibodies be utilized to investigate the opposing roles of Granzymes A and B in immune responses?

Recent research has revealed that Granzymes A and B can have opposing effects in certain immune contexts. To investigate this phenomenon:

• Multiplex immunofluorescence:

  • Implement simultaneous staining for GZMA and GZMB using differentially labeled antibodies

  • Analyze co-expression patterns in various immune cell populations

  • Correlate with functional markers (activation, exhaustion)

• Infection models:

  • Use GZMB antibodies alongside GZMA detection in models where differential effects have been observed

  • For example, in Salmonella infection models, where GZMA appears protective while GZMB may exacerbate disease

  • Analyze tissue and cellular distribution patterns in wild-type versus knockout models

• Cytokine correlation studies:

  • Combine GZMB antibody staining with cytokine detection

  • Analyze how GZMB positivity correlates with pro-inflammatory or anti-inflammatory cytokine profiles

  • Compare these patterns with GZMA-expressing populations

• Single-cell analysis:

  • Apply GZMB antibodies in single-cell protein analysis platforms

  • Correlate with transcriptomic data to understand differential regulation

  • Identify cell populations with unique GZMA/GZMB expression ratios .

Why might I observe inconsistent GZMB staining in my experiments, and how can I address this?

Inconsistent GZMB staining can result from several factors:

• Post-translational regulation:

  • GZMB is subject to complex post-transcriptional regulation

  • Transcript levels may not correlate with protein expression

  • Solution: Compare results from protein detection (antibody-based) with transcript analysis

• Antibody epitope accessibility:

  • GZMB storage in granules may limit epitope accessibility

  • Solution: Optimize permeabilization conditions; consider stronger detergents for granule disruption

• Variable activation states:

  • Resting cells may have minimal GZMB expression

  • Solution: Standardize activation protocols; consider time-course experiments to capture expression dynamics

• Protein degradation:

  • GZMB, as a protease, can be unstable in certain preparations

  • Solution: Include protease inhibitors in all sample preparation steps; process samples consistently and rapidly

• Technical variability:

  • Antibody lot-to-lot variation can affect staining intensity

  • Solution: Validate each new antibody lot; include consistent positive controls with known staining patterns .

How should I interpret GZMB antibody data in the context of conflicting functional studies?

When interpreting GZMB antibody data in light of functional discrepancies:

• Context-dependent functions:

  • GZMB may have different effects depending on microenvironment and cellular source

  • Solution: Carefully document experimental context; include detailed methods descriptions

• Species differences:

  • Human and mouse GZMB may have distinct functions

  • Solution: Avoid direct cross-species extrapolation; validate findings in relevant species models

• Compensatory mechanisms:

  • In knockout models, other granzymes may compensate for GZMB loss

  • Solution: Consider using multiple granzyme detection methods; validate with inhibitors or knockdown approaches

• Canonical vs. non-canonical functions:

  • GZMB detection doesn't distinguish between cytotoxic and non-cytotoxic roles

  • Solution: Combine GZMB antibody data with functional readouts specific to each hypothesized function

• Artifact vs. biological significance:

  • Strong GZMB expression doesn't necessarily indicate functional relevance

  • Solution: Validate antibody findings with independent functional assays; include appropriate controls .

What controls should be implemented when using GZMB antibodies to ensure result reliability?

Rigorous controls are essential for reliable GZMB antibody-based studies:

• Positive controls:

  • Include known GZMB-expressing samples (activated NK cells, CTLs)

  • Use cell lines with confirmed GZMB expression (NK-92, activated primary T cells)

• Negative controls:

  • Include samples known to lack GZMB expression

  • Use isotype control antibodies at equivalent concentrations

  • Consider GZMB knockout or knockdown samples when available

• Specificity controls:

  • Pre-adsorption with recombinant GZMB protein

  • Use multiple antibodies targeting different GZMB epitopes

  • Include western blot validation alongside other applications

• Technical controls:

  • Antibody titration to determine optimal concentration

  • Implement fluorescence minus one (FMO) controls in flow cytometry

  • Include secondary antibody-only controls in indirect detection methods

• Validation across methods:

  • Confirm key findings with orthogonal techniques (e.g., mass spectrometry)

  • Correlate protein detection with mRNA analysis while acknowledging potential discrepancies .

How can GZMB antibodies contribute to understanding the role of extracellular Granzyme B in disease pathogenesis?

Extracellular GZMB is increasingly recognized as a mediator in various pathologies:

• Tissue section analysis:

  • Use GZMB antibodies to detect extracellular GZMB deposits in tissues

  • Implement dual staining with extracellular matrix components

  • Apply confocal microscopy to distinguish membrane-bound from truly extracellular GZMB

• Body fluid analysis:

  • Develop or utilize ELISA-based assays using GZMB antibodies for quantification in serum, synovial fluid, or other biological fluids

  • Correlate levels with disease severity and progression

• Ex vivo degradation models:

  • Apply recombinant GZMB to tissue sections or matrices

  • Use antibodies to detect resulting cleavage products or altered matrix architecture

  • Implement in disease-specific contexts (e.g., cardiovascular tissue, skin)

• Therapeutic potential assessment:

  • Use antibodies to evaluate the efficacy of GZMB inhibitors

  • Monitor extracellular GZMB levels during treatment courses

  • Develop potential neutralizing antibodies against extracellular GZMB .

What methodological approaches can combine GZMB antibody detection with functional analysis of cytotoxic immune responses?

Integrating GZMB detection with functional cytotoxicity requires sophisticated approaches:

• Live cell imaging:

  • Utilize non-disruptive GZMB antibody fragments or nanobodies

  • Combine with target cell death indicators

  • Track GZMB release and subsequent target cell effects in real-time

• Microfluidic systems:

  • Apply GZMB antibodies in chip-based cytotoxicity assays

  • Monitor GZMB release kinetics in relation to target cell killing

  • Analyze at single-cell resolution

• Mass cytometry (CyTOF):

  • Incorporate metal-conjugated GZMB antibodies in panels with functional readouts

  • Analyze high-dimensional data to correlate GZMB expression with killing capacity

  • Identify novel cell subsets with unique functional profiles

• In situ analysis of immune synapses:

  • Use high-resolution microscopy with GZMB antibodies

  • Examine GZMB polarization and release at the immunological synapse

  • Correlate with structural and functional markers of synapse formation .

How might GZMB antibodies be utilized to investigate the role of Granzyme B in non-immune cell populations?

As GZMB expression expands beyond classical immune cells, novel applications emerge:

• Lineage-specific analysis:

  • Apply GZMB antibodies alongside cell-type specific markers

  • Investigate conditions that induce GZMB in non-immune cells

  • Study regulatory mechanisms in diverse cell types

• Developmental biology applications:

  • Track GZMB expression during tissue development and remodeling

  • Analyze potential roles in programmed cell death during development

  • Implement lineage tracing with GZMB detection

• Pathology applications:

  • Examine GZMB expression in fibrotic disorders

  • Analyze vascular remodeling in relation to GZMB+ cells

  • Investigate potential roles in wound healing and tissue repair

• Single-cell proteomics:

  • Include GZMB antibodies in single-cell protein profiling

  • Identify rare GZMB-expressing populations

  • Correlate with functional states and disease progression .