GSDMB Antibody

What is GSDMB and what cellular functions has it been implicated in?

Gasdermin B (GSDMB) is a member of the gasdermin family of proteins, which were initially characterized for their role in pyroptosis, a form of programmed cell death. GSDMB is currently the least characterized member of this family . Recent research has demonstrated GSDMB's involvement in several critical cellular processes:

• Epithelial restitution and repair: GSDMB appears to play a role in mucosal healing, particularly in intestinal epithelium, with elevated expression in restituting epithelium overlying areas of active inflammation .

• Cell migration: In cancer contexts, GSDMB promotes cell migration and metastatic behavior .

• Therapy resistance: GSDMB overexpression has been linked to resistance against targeted therapies, particularly in HER2-positive breast cancers .

Unlike other gasdermin family members, GSDMB doesn't appear to induce cell death through the canonical pyroptotic pathway in most contexts, suggesting distinct biological functions .

What are the most validated applications for GSDMB antibodies in research?

Based on published research and commercial antibody validation data, GSDMB antibodies have been successfully employed in multiple applications:

Different antibody clones may perform better in specific applications, so researchers should review validation data when selecting an antibody for their particular experimental system .

What is the molecular structure and observed weight of the GSDMB protein?

The GSDMB protein has:

• Calculated molecular weight: 47 kDa based on its 411 amino acid sequence

• Observed molecular weight: Typically observed at 45-50 kDa in Western blot applications

• UniProt ID: Q8TAX9

• GenBank Accession Number: BC025682

The protein has distinct functional domains common to gasdermins, though its molecular activity differs from better-characterized family members like GSDMD .

How do GSDMB expression patterns differ across cell types and disease states?

GSDMB expression varies significantly across tissues and in disease states:

• Healthy tissues: Survey of 27 different healthy tissues shows greatest GSDMB abundance in gastrointestinal-associated organs (stomach, small intestine, colon) with predominant expression in epithelial cells .

• Inflammatory Bowel Disease (IBD):

  • Dramatic increases (5.09-fold and 5.83-fold) in GSDMB expression in freshly isolated intestinal epithelial cells from non-resolving Crohn's Disease and Ulcerative Colitis patients compared to healthy controls .

  • Single-cell RNA sequencing reveals differential distribution of GSDMB among intestinal epithelial cell subtypes, predominantly in colonocytes, crypt top colonocytes, and to a lesser extent, goblet cells .

• Cancer contexts:

  • Overexpression/amplification occurs in approximately 60% of HER2-positive breast cancers .

  • Expression has been documented in colorectal cancer, though its prognostic significance is still being established .

What methodological approaches can be used to study GSDMB's role in cell death mechanisms?

While GSDMB belongs to the gasdermin family, its role in cell death appears distinct from the canonical pyroptotic pathway. Researchers can investigate this using:

• Cell death assays:

  • SYTOX™ deep red staining to assess membrane permeability

  • LDH release assays using kits such as the Cyto Tox 96 Non-Radioactive Cytotoxicity Assay

  • Analysis of cleaved caspases (caspase 1, 8, 3, and 7) by immunoblotting to determine if GSDMB activates specific death pathways

• Genetic manipulation:

  • GSDMB knockout models (GSDMB−/−) have been developed to compare with wild-type under various stimulation conditions

  • GSDMB overexpression models to assess effects on cell survival and death pathways

• Mechanistic studies:

  • Immunoprecipitation to identify GSDMB binding partners

  • Subcellular localization studies using confocal microscopy to determine compartmentalization

Research has shown that unlike GSDMD-dependent activation of inflammasomes (which triggers significant cell death), GSDMB may have different functions under similar stimulation conditions .

What are the current approaches for targeting GSDMB in disease models?

Several innovative approaches have been developed to target GSDMB in disease models:

• Antibody-based nanomedicine:

  • Hyaluronic acid-biocompatible nanocapsules have been developed to deliver anti-GSDMB antibodies intracellularly in HER2 breast cancer cells .

  • This approach has shown promising results:

    • Reduced in vitro cell migration induced by GSDMB

    • Enhanced sensitivity to trastuzumab therapy

    • Reduced tumor growth by increasing apoptotic rate in orthotopic breast cancer xenografts

    • Diminished lung metastasis in vivo

• Mechanistic targeting:

  • Anti-GSDMB antibodies can increase GSDMB binding to sulfatides, decreasing migratory behavior and potentially upregulating intrinsic pro-cell death activity .

• Combination approaches:

  • Studies have explored autophagy inhibition in conjunction with targeting GSDMB to overcome therapy resistance in HER2-positive cancers .

What are the optimal protocols for GSDMB detection in tissue samples?

For successful detection of GSDMB in tissue samples, researchers should consider these validated protocols:

For Immunohistochemistry (IHC):

• Antigen retrieval: Two effective methods have been documented:

  • TE buffer at pH 9.0 (preferred method for many applications)

  • Citrate buffer at pH 6.0 as an alternative

• Antibody dilution range: 1:50-1:500 is typically recommended, though optimization for specific tissue types is advised

• Detection system: A two-step method (such as PV-9000 Polymer Detection System) with DAB solution for color rendering followed by hematoxylin counterstaining has been successfully used

For Immunofluorescence (IF):

• Fixation: Standard paraformaldehyde fixation

• Dilution range: 1:400-1:1600, with optimization for specific cell types

• Controls: Include GSDMB knockout or knockdown samples where possible

How should researchers quantify and interpret GSDMB expression patterns in clinical samples?

Quantification of GSDMB expression in clinical samples requires attention to multiple parameters:

• Subcellular localization scoring:

  • GSDMB expression should be evaluated separately in the membrane, cytoplasm, and nucleus as different localization patterns may have distinct biological significance .

  • Digital scanning of slides (using systems like NanoZoomer 2.0 HT) enables more precise quantification .

• Immune cell evaluation:

  • Consider GSDMB expression not only in target cells (e.g., epithelial or cancer cells) but also in immune cells within the microenvironment .

  • Double immunofluorescence staining can be used to identify specific GSDMB-positive immune cell populations (T cells, B cells, macrophages) .

• Correlation analysis:

  • GSDMB expression should be correlated with relevant clinical parameters and other molecular markers (e.g., CD3+, CD4+, CD8+ T lymphocytes, CD20+ B lymphocytes, CD68+ macrophages, and S100A8+ immune cells in cancer contexts) .

  • Univariate and multivariate survival analyses provide insights into prognostic significance .

What controls and validation steps are essential when using GSDMB antibodies?

To ensure reliable and reproducible results with GSDMB antibodies, include these validation steps:

• Positive controls:

  • Cell lines with confirmed GSDMB expression: MCF-7, COLO 320, PC-3, and A431 cells have been validated for Western blot .

  • Tissue types: Human liver cancer and colon cancer tissues have shown reliable GSDMB staining patterns .

• Negative controls:

  • Use PBS buffer instead of primary antibody to assess non-specific binding .

  • When possible, include GSDMB knockout or knockdown samples .

• Antibody validation:

  • Western blotting to confirm antibody specificity at the expected molecular weight (45-50 kDa) .

  • Comparison of multiple antibody clones for your specific application and cell/tissue type .

  • Titration of antibody concentration to determine optimal signal-to-noise ratio .

How can GSDMB antibodies be used to investigate inflammatory bowel disease mechanisms?

GSDMB antibodies have proven valuable in elucidating IBD mechanisms:

• Tissue expression patterns:

  • IHC and IF studies have revealed GSDMB expression in restituting epithelium overlying areas of active inflammation in IBD tissues .

  • Co-localization studies using GSDMB antibodies (red) with epithelial-specific cell surface markers like EpCAM (green) confirm expression along the plasma membrane in inflamed IBD tissues .

• Single-cell analysis integration:

  • GSDMB antibodies can be used to validate findings from single-cell RNA sequencing data that show differential distribution of GSDMB among intestinal epithelial cell subtypes in IBD .

• Functional studies:

  • Combining GSDMB antibody staining with cell death markers helps distinguish GSDMB's role in repair versus pyroptosis .

  • Gene ontology analysis of GSDMB-expressing cells has identified upregulated biological processes, providing targets for further antibody-based validation .

What is the significance of GSDMB in breast cancer research, and how can antibodies help characterize its role?

GSDMB has emerged as a significant factor in breast cancer, particularly HER2-positive subtypes:

• Expression correlation with cancer progression:

  • GSDMB overexpression/amplification occurs in approximately 60% of HER2-positive breast cancers .

  • Antibody-based detection methods (IHC, WB) can identify patients with GSDMB overexpression who might benefit from targeted approaches .

• Mechanistic studies:

  • Anti-GSDMB antibodies delivered via nanocapsules have demonstrated the ability to:

    • Reduce in vitro cell migration induced by GSDMB

    • Enhance sensitivity to trastuzumab (a standard HER2-targeted therapy)

    • Reduce tumor growth by increasing apoptotic rate in breast cancer xenografts

    • Diminish lung metastasis in vivo

• Resistance mechanisms:

  • Antibody-based studies have helped decode GSDMB's role in therapy resistance, revealing:

    • Potential involvement in autophagy regulation

    • Mechanism through which GSDMB binding to sulfatides affects cell behavior

    • Changes in GSDMB-protein interactions during therapy resistance

What are common challenges in GSDMB antibody experiments and how can they be addressed?

Researchers may encounter several challenges when working with GSDMB antibodies:

• Variability in subcellular localization:

  • GSDMB can be detected in multiple cellular compartments (membrane, cytoplasm, nucleus) .

  • Solution: Perform subcellular fractionation before Western blotting or use high-resolution imaging to clarify localization patterns.

• Tissue disintegration during IHC:

  • Some tissues may disintegrate during processing, resulting in no obtainable data .

  • Solution: Optimize fixation times and consider using tissue microarrays for initial screening before proceeding to whole-tissue slides .

• Antibody clone selection:

  • Different clones may perform better in specific applications.

  • Solution: When possible, test multiple antibodies (monoclonal vs. polyclonal) for your specific application. The search results mention several validated options with specific catalog numbers (67527-1-Ig, 12885-1-AP, ab215729) .

How can researchers optimize GSDMB antibody protocols for novel tissue types or experimental systems?

When adapting GSDMB antibody protocols to new experimental systems:

• Dilution optimization:

  • Start with the manufacturer's recommended range (e.g., 1:500-1:1000 for WB, 1:50-1:500 for IHC) .

  • Perform a dilution series to determine optimal concentration for your specific tissue/cell type.

• Antigen retrieval method testing:

  • Compare both recommended methods: TE buffer (pH 9.0) and citrate buffer (pH 6.0) .

  • Evaluate signal intensity and background for each method.

• Storage and handling:

  • Follow recommended storage conditions (typically -20°C with 0.02% sodium azide and 50% glycerol at pH 7.3) .

  • Aliquot antibodies to avoid freeze-thaw cycles.

  • Consider stability period (typically stable for one year after shipment when properly stored) .

What advanced techniques can be combined with GSDMB antibodies for comprehensive functional studies?

Researchers can enhance GSDMB studies by combining antibody detection with:

• Double immunofluorescence:

  • Has been successfully used to identify GSDMB-expressing immune cell populations .

  • Enables co-localization studies with other markers of interest.

• Proximity ligation assays:

  • Can identify protein-protein interactions involving GSDMB.

  • Particularly valuable for studying GSDMB's interactions with other cellular components.

• Live-cell imaging:

  • When combined with appropriate tags or antibody fragments, can monitor GSDMB dynamics.

  • Useful for studying trafficking and subcellular localization changes in response to stimuli.

• Targeted delivery systems:

  • Hyaluronic acid-based nanocapsules have been used to deliver anti-GSDMB antibodies intracellularly .

  • This approach enables both research applications and potential therapeutic development.

What emerging applications of GSDMB antibodies are being explored in current research?

Several innovative applications for GSDMB antibodies are emerging:

• Therapeutic antibody delivery:

  • Intracellular delivery of anti-GSDMB antibodies via nanocapsules represents a new targeted therapeutic strategy for aggressive HER2 cancers with poor prognosis .

  • This approach has shown efficacy in reducing diverse protumor GSDMB functions including migration, metastasis, and therapy resistance .

• Biomarker development:

  • Correlation of GSDMB staining patterns with prognosis in colorectal cancer and other malignancies .

  • Integration with immune cell markers to characterize the tumor microenvironment .

• Mechanism exploration:

  • Investigating GSDMB's role in the crosstalk between different cell death pathways (pyroptosis, necroptosis) .

  • Understanding how GSDMB contributes to therapy resistance mechanisms, particularly in HER2-targeted cancer treatments .