GSDMA Antibody, Biotin conjugated

What is GSDMA and what cellular functions does it regulate?

GSDMA belongs to the gasdermin family of proteins, which comprises five members (GSDMA-GSDME) in humans. These proteins are predominantly expressed in the skin, mucosa, and immune sentinel cells and function as key executioners of inflammatory cell death, specifically pyroptosis . GSDMA is particularly noted for its role in inducing apoptosis . Gasdermins are pore-forming proteins that, upon activation, create membrane pores leading to cellular swelling, membrane rupture, and release of cellular contents including pro-inflammatory cytokines. This process recruits immune cells to infection sites and promotes protective immunity . Unlike some other gasdermin family members such as GSDMD, which has been specifically demonstrated to provide protection against oral Salmonella infection , the precise physiological and pathological roles of GSDMA continue to be an active area of investigation.

What are the key characteristics of biotin-conjugated GSDMA antibodies?

Biotin-conjugated GSDMA polyclonal antibodies, such as the bs-16331r-biotin variant, are generated from rabbits immunized with KLH-conjugated synthetic peptides derived from human GSDMA . These antibodies typically:

• Target specific epitopes within the GSDMA protein (immunogen range often between amino acids 21-120 of the 445 amino acid protein)

• Demonstrate reactivity with mouse and rat GSDMA, with predicted cross-reactivity to human, dog, cow, and horse GSDMA proteins

• Maintain a concentration of approximately 1μg/μl

• Are stored in aqueous buffered solutions containing TBS (pH 7.4) with BSA, preservatives, and glycerol

• Require storage at -20°C for optimal stability (shelf life of approximately 12 months)

• Have applications in Western Blotting, ELISA, and immunohistochemistry techniques

The biotin conjugation provides significant advantages for detection sensitivity and versatility when used with avidin/streptavidin detection systems, making these antibodies particularly valuable for complex tissue analysis or low-abundance protein detection scenarios.

What are the optimal dilution ratios for different experimental applications of biotin-conjugated GSDMA antibodies?

The optimal antibody dilution varies significantly depending on the specific application, sample type, and detection method. Based on technical specifications for GSDMA antibodies, the following dilution ranges are recommended:

How should researchers design experiments to study GSDMA's role in pyroptosis using biotin-conjugated antibodies?

When investigating GSDMA's role in pyroptosis using biotin-conjugated antibodies, consider this methodological approach:

• Experimental Design:

  • Use appropriate cell models that express GSDMA (skin epithelial cells, certain immune cells)

  • Include both positive controls (cells known to express GSDMA) and negative controls (GSDMA-knockout cells or tissues)

  • Design time-course experiments to capture the dynamic process of pyroptosis

• Induction of Pyroptosis:

  • Treat cells with known pyroptosis inducers (e.g., canonical inflammasome activators)

  • Monitor morphological changes characteristic of pyroptosis (cell swelling, membrane rupture)

• Detection Methods:

  • Immunofluorescence with biotin-conjugated GSDMA antibodies (1:100-200 dilution) followed by streptavidin-conjugated fluorophores to visualize GSDMA localization before and during pyroptosis

  • Western blotting to detect GSDMA processing (look for both full-length ~49 kDa and cleaved N-terminal fragments)

  • Flow cytometry to quantify cells undergoing pyroptosis in correlation with GSDMA expression

  • Co-localization studies with membrane markers to assess pore formation

• Functional Validation:

  • Compare wild-type and GSDMA-knockdown/knockout models

  • Measure pyroptosis-associated outcomes: LDH release, IL-1β secretion, propidium iodide uptake

  • Assess downstream effects on immune cell recruitment using migration assays

This comprehensive approach allows for thorough characterization of GSDMA's role in pyroptotic cell death while leveraging the enhanced sensitivity provided by biotin-conjugated antibodies.

How can biotin-conjugated GSDMA antibodies be utilized to investigate cross-talk between gasdermin family members?

Investigating gasdermin family cross-talk requires sophisticated experimental approaches. Biotin-conjugated GSDMA antibodies offer several methodological advantages:

• Multiplex Immunofluorescence Analysis:

  • Utilize biotin-conjugated GSDMA antibodies in combination with directly-labeled antibodies against other gasdermin family members

  • Employ streptavidin conjugated to spectrally distinct fluorophores (e.g., far-red) to differentiate GSDMA from other gasdermins labeled with green or red fluorophores

  • This approach allows simultaneous visualization of multiple gasdermin family members within the same cellular compartments

• Sequential Immunoprecipitation Strategy:

  • First, immunoprecipitate with biotin-conjugated GSDMA antibodies using streptavidin beads

  • Analyze precipitates for co-precipitating gasdermin family members by Western blotting

  • Follow with reverse co-immunoprecipitation using antibodies against other gasdermins

  • This reciprocal approach helps validate authentic protein-protein interactions

• Proximity Ligation Assays (PLA):

  • Combine biotin-conjugated GSDMA antibodies with primary antibodies against other gasdermin family members

  • Use streptavidin-conjugated PLA probes together with secondary antibody-conjugated PLA probes

  • This method generates fluorescent signals only when proteins are in close proximity (<40 nm), providing spatial evidence of potential interactions

• Functional Cross-talk Analysis:

  • In models with selective knockdown of individual gasdermins, use biotin-conjugated GSDMA antibodies to assess whether GSDMA localization, processing, or activity changes when other family members are absent

  • Employ purified recombinant gasdermins with biotin-labeled GSDMA to study direct interactions in cell-free systems

These approaches can reveal whether different gasdermin family members work cooperatively, competitively, or independently in executing pyroptosis across different cellular contexts and inflammatory stimuli.

What are the critical considerations when interpreting co-localization data from immunohistochemistry using biotin-conjugated GSDMA antibodies?

When analyzing co-localization data from immunohistochemistry experiments using biotin-conjugated GSDMA antibodies, researchers should address several critical considerations:

What strategies can resolve high background issues when using biotin-conjugated GSDMA antibodies in immunohistochemistry?

High background is a common challenge when using biotin-conjugated antibodies in immunohistochemistry. To resolve this issue with GSDMA biotin-conjugated antibodies, consider implementing these methodological approaches:

• Address Endogenous Biotin Interference:

  • Implement an avidin-biotin blocking step before primary antibody incubation

  • Commercially available kits provide sequential application of avidin (binds endogenous biotin) followed by biotin (saturates remaining avidin binding sites)

  • This is particularly crucial for biotin-rich tissues such as liver, kidney, and brain

• Optimize Blocking Protocol:

  • Extend blocking time to 1-2 hours at room temperature

  • Use a combination of normal serum (5-10%) from the species of the secondary antibody

  • Add 0.1-0.3% Triton X-100 for better penetration in fixed tissues

  • Include 1% BSA to reduce nonspecific protein interactions

  • Consider specialized blocking reagents like Mouse-on-Mouse blocking solutions if using mouse tissues

• Antibody Dilution and Incubation Adjustments:

  • Test a broader dilution series beyond the recommended 1:200-400 range for IHC-P

  • Increase antibody dilution gradually if background persists

  • Switch to overnight incubation at 4°C instead of shorter room temperature incubation

  • Implement additional washing steps (at least 3 x 10 minutes) with gentle agitation

• Detection System Modifications:

  • Try different streptavidin conjugates if one gives high background

  • Consider using streptavidin conjugates with smaller reporter molecules

  • Dilute the streptavidin conjugate further than recommended

  • Use tyramide signal amplification only when absolutely necessary for low-abundance targets, as it can amplify background

  • Consider alternative detection methods if biotin-based systems consistently yield high background

Employing these strategies systematically while changing one variable at a time will help identify the source of background issues and optimize protocols for clean, specific staining with biotin-conjugated GSDMA antibodies.

How should researchers troubleshoot discrepancies between Western blot and immunohistochemistry results when using biotin-conjugated GSDMA antibodies?

When faced with discrepancies between Western blot (WB) and immunohistochemistry (IHC) results using biotin-conjugated GSDMA antibodies, researchers should systematically investigate several potential explanations:

• Epitope Accessibility Differences:

  • In WB, proteins are denatured, exposing all epitopes

  • In IHC, epitopes may be partially masked by protein folding or interactions

  • Solution: Try different antigen retrieval methods for IHC (heat-induced vs. enzymatic)

  • Test multiple GSDMA antibodies targeting different epitopes (N-terminal vs. C-terminal regions)

• Protein Modification Status:

  • Post-translational modifications may differ between lysed samples and fixed tissues

  • Phosphorylation, cleavage, or complex formation can affect antibody recognition

  • Solution: Use phosphatase treatment on some samples to assess impact of phosphorylation

  • Compare native vs. reducing conditions in WB to evaluate complex formation

• Cross-Reactivity Profiles:

  • Create a validation table comparing reactivity across applications:

  Sample Type	Expected MW	WB Result	IHC Result	Possible Explanation
  Mouse liver	49 kDa	Positive	Negative	Epitope masking in tissue
  Human skin	49 kDa	Weak band	Strong signal	Enrichment in specific cell types
  COLO 320 cells	49 kDa	Multiple bands	Diffuse staining	Potential degradation products

• Methodological Troubleshooting:

  • For WB discrepancies:

    • Verify protein transfer efficiency with reversible staining

    • Try gradient gels to better resolve GSDMA (49 kDa)

    • Increase blocking stringency to reduce non-specific bands

    • Use positive control lysates known to express GSDMA

  • For IHC discrepancies:

    • Optimize fixation time (overfixation can mask epitopes)

    • Try fresh frozen sections if formalin-fixed samples give poor results

    • Employ signal amplification systems for low abundance detection

    • Test biotin blocking systems to reduce background

• Biological Context Considerations:

  • GSDMA expression may be restricted to specific cell types within tissues

  • Subcellular localization changes during pyroptosis activation

  • Solution: Use cell type-specific markers in parallel with GSDMA staining

  • Perform fractionation experiments to track GSDMA in different cellular compartments

By systematically addressing these variables, researchers can better understand the source of discrepancies and determine which application provides the most reliable data for their specific research question.

How can researchers design experiments to investigate GSDMA's role in disease models using biotin-conjugated antibodies?

Designing experiments to investigate GSDMA's role in disease models requires a comprehensive approach leveraging the advantages of biotin-conjugated antibodies:

• Disease Model Selection and Validation:

  • Choose models relevant to GSDMA's known expression pattern (epithelial tissues, skin, mucosa)

  • Consider models of inflammatory conditions, epithelial barrier dysfunction, or cancer

  • Validate GSDMA expression in your model system before extensive experimentation

  • Compare GSDMA expression patterns between healthy and diseased states using biotin-conjugated antibodies at 1:200-400 dilution for IHC-P

• Multi-parametric Analysis Strategy:

  • Implement sequential tissue staining protocols:

    • First round: Biotin-conjugated GSDMA antibody with streptavidin-fluorophore detection

    • Second round: Antibodies against disease markers or immune cell populations

    • Digital image analysis to correlate GSDMA localization with disease progression

  • Combine with laser capture microdissection to isolate GSDMA-positive regions for molecular analysis

• Functional Assessment Methods:

  • Genetic approaches:

    • Generate conditional GSDMA knockout models using Cre-lox system

    • Employ CRISPR/Cas9 for cell line modifications

    • Use inducible overexpression systems to study dosage effects

  • Pharmacological approaches:

    • Test pyroptosis inhibitors in disease models

    • Assess effects on GSDMA processing and localization

  • Real-time monitoring:

    • Employ live-cell imaging with membrane integrity markers

    • Measure inflammatory cytokine release in correlation with GSDMA activation

• Translational Research Considerations:

  • For potential clinical correlations:

    • Apply biotin-conjugated GSDMA antibodies to patient-derived xenografts

    • Analyze GSDMA expression in tissue microarrays of patient samples

    • Correlate GSDMA expression patterns with clinical outcomes

  • Compare with other gasdermin family members to assess potential redundancy or specialized functions, similar to approaches used in studying GSDMD's protective role against Salmonella infection

• Controls and Validation Framework:

  • Positive controls: Include tissues known to express GSDMA (stomach, skin)

  • Negative controls: Use tissues from GSDMA-knockout models

  • Antibody validation: Confirm specificity through peptide competition assays

  • Independent verification: Validate key findings with alternative detection methods beyond biotin-streptavidin systems

This comprehensive experimental framework allows researchers to thoroughly investigate GSDMA's role in disease pathophysiology while maximizing the detection sensitivity offered by biotin-conjugated antibodies.

What are the important considerations when using biotin-conjugated GSDMA antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence studies with biotin-conjugated GSDMA antibodies require careful experimental design and execution:

By addressing these considerations systematically, researchers can obtain reliable multiplex data that reveals the complex relationships between GSDMA and other components of pyroptotic pathways across different biological contexts.

How might biotin-conjugated GSDMA antibodies contribute to understanding gasdermin evolutionary relationships across species?

Biotin-conjugated GSDMA antibodies can serve as valuable tools for comparative studies investigating gasdermin evolution across species. This research direction involves several methodological considerations:

• Cross-Species Reactivity Assessment:

  • Systematic testing of biotin-conjugated GSDMA antibodies across tissues from different species:

    • Mammalian models (mouse, rat, human, non-human primates)

    • Non-mammalian vertebrates (zebrafish, chickens)

    • Evolutionary distant organisms where gasdermin homologs are predicted

  • Create a comprehensive cross-reactivity profile based on epitope conservation:

  Species	GSDMA Homolog	Epitope Conservation	Reactivity with Anti-human GSDMA
  Human	GSDMA	100% (reference)	Strong (WB, IHC)
  Mouse	Gsdma1-3	78-85%	Moderate (WB, IHC)
  Rat	Gsdma	76%	Moderate (WB)
  Dog	GSDMA	80% (predicted)	Weak (predicted)
  Zebrafish	gsdmea	<60%	Not expected

• Structural and Functional Conservation Analysis:

  • Immunoprecipitation with biotin-conjugated antibodies followed by mass spectrometry to identify species-specific binding partners

  • Comparative immunohistochemistry to map expression patterns across homologous tissues

  • Functional assays to assess conservation of pore-forming activity:

    • Liposome leakage assays with immunopurified GSDMA from different species

    • Cell permeabilization studies in heterologous expression systems

• Evolutionary Divergence Mapping:

  • Epitope mapping using peptide arrays to identify conserved and divergent regions

  • Correlation of antibody reactivity with phylogenetic distances

  • Investigation of selective pressures (positive/negative selection) on specific GSDMA domains

  • Comparative analysis with other gasdermin family members (GSDMB, GSDMD) to understand family evolution

• Technical Advantages of Biotin Conjugation:

  • Enhanced sensitivity for detecting low-abundance homologs

  • Versatility across detection platforms

  • Potential for retrieving gasdermin-containing complexes for evolutionary proteomics

  • Compatibility with archived/fixed specimens from diverse species

• Experimental Design Considerations:

  • Include antibodies targeting highly conserved regions (e.g., N-terminal pore-forming domain)

  • Employ recombinant GSDMA proteins as calibration standards

  • Use genetic models (e.g., GSDMA-deficient mice) to validate specificity

  • Consider potential cross-reactivity with other gasdermin family members

This evolutionary approach could reveal insights into how pyroptotic mechanisms evolved across species and help identify conserved functional domains that might represent targets for therapeutic intervention in gasdermin-mediated diseases.

What methodological approaches can researchers use to study the interaction between GSDMA and lipid membranes using biotin-conjugated antibodies?

Investigating GSDMA-membrane interactions presents unique challenges that require specialized approaches leveraging biotin-conjugated antibodies:

• In Vitro Membrane System Analyses:

  • Liposome binding assays:

    • Prepare liposomes with varying lipid compositions

    • Incubate with recombinant GSDMA or cell lysates

    • Detect bound GSDMA using biotin-conjugated antibodies and streptavidin-fluorophores

    • Quantify binding by fluorescence intensity measurements

  • Supported lipid bilayer (SLB) systems:

    • Create SLBs on glass surfaces with defined composition

    • Visualize GSDMA binding and oligomerization using total internal reflection fluorescence (TIRF) microscopy

    • Use biotin-conjugated antibodies with quantum dot-conjugated streptavidin for single-molecule tracking

• Cellular Membrane Interaction Studies:

  • Live-cell surface labeling:

    • Use non-permeabilizing conditions to detect plasma membrane-associated GSDMA

    • Apply biotin-conjugated antibodies to live cells followed by streptavidin-fluorophore detection

    • Compare surface labeling before/after pyroptosis induction

  • Membrane fractionation approach:

    • Separate cellular membranes through ultracentrifugation

    • Analyze GSDMA distribution across fractions by Western blotting

    • Correlate with membrane lipid composition analysis

• Super-resolution Microscopy Techniques:

  • STORM/PALM imaging:

    • Use biotin-conjugated GSDMA antibodies with photoswitchable fluorophore-conjugated streptavidin

    • Visualize nanoscale organization of GSDMA pores in membranes

    • Achieve 10-20 nm resolution of GSDMA structures

  • Correlative light-electron microscopy:

    • Locate GSDMA using biotin-conjugated antibodies and gold-conjugated streptavidin

    • Correlate with membrane ultrastructure at electron microscopy level

• Lipid Interaction Specificity Assessment:

  • Lipid strip/array binding assays:

    • Incubate recombinant GSDMA with membranes containing spotted lipids

    • Detect binding using biotin-conjugated antibodies

    • Compare binding profiles with other gasdermin family members

  • Functional relevance to pore formation:

    • Analyze how specific lipid interactions correlate with pore-forming activity

    • Compare results with findings from other gasdermins like GSDMD, which has been shown to provide protection against bacterial infection through pore formation

• Advanced Biophysical Approaches:

  • Surface plasmon resonance (SPR):

    • Immobilize lipid membranes on sensor chips

    • Measure GSDMA binding kinetics in real-time

    • Validate specificity using biotin-conjugated antibodies

  • Atomic force microscopy (AFM):

    • Visualize GSDMA-induced membrane pores at nanometer resolution

    • Correlate with immunolabeling using biotin-conjugated antibodies

    • Measure mechanical properties of GSDMA-altered membranes

These methodological approaches provide complementary insights into the mechanisms by which GSDMA interacts with and potentially disrupts cellular membranes, advancing our understanding of its role in pyroptotic cell death pathways.

How can biotin-conjugated GSDMA antibodies be integrated into high-throughput screening approaches for drug discovery?

Biotin-conjugated GSDMA antibodies offer unique advantages for high-throughput screening (HTS) in drug discovery focused on pyroptosis modulation:

• Assay Development for Primary Screening:

  • Cell-based ELISA approaches:

    • Seed cells in 384-well plates and treat with compound libraries

    • Fix and permeabilize cells at various timepoints

    • Detect GSDMA cleavage or membrane translocation using biotin-conjugated antibodies

    • Quantify using HRP-streptavidin and colorimetric/chemiluminescent substrates

  • Automated immunofluorescence platforms:

    • Employ biotin-conjugated GSDMA antibodies with fluorescent streptavidin conjugates

    • Multiplex with markers for cell viability and membrane integrity

    • Analyze using high-content imaging systems for multiple parameters:

      • GSDMA subcellular localization

      • Evidence of pore formation

      • Cell morphological changes

      • Quantitative intensity measurements

• Hit Validation Methodologies:

  • Dose-response characterization:

    • Test hits across concentration ranges (nM to μM)

    • Quantify GSDMA cleavage, oligomerization, and membrane translocation

    • Establish EC50/IC50 values for GSDMA modulation

  • Mechanism of action studies:

    • Immunoprecipitation using biotin-conjugated GSDMA antibodies to assess compound effects on protein interactions

    • In-cell target engagement assays to confirm direct binding to GSDMA

    • Counter-screening against other gasdermin family members to assess selectivity

• Advanced Screening Applications:

  • FRET-based assays:

    • Design systems using biotin-conjugated GSDMA antibodies paired with fluorophore-labeled streptavidin

    • Monitor conformational changes or protein-protein interactions in real-time

    • Enables kinetic measurements suitable for HTS platforms

  • Surface plasmon resonance screening:

    • Immobilize biotin-conjugated GSDMA antibodies on streptavidin sensor chips

    • Screen for compounds that alter GSDMA binding to membrane components

    • Provides binding kinetics data for hit compounds

• Translational Research Integration:

  • Patient-derived cell screening:

    • Apply biotin-conjugated GSDMA antibody assays to primary cells from patients

    • Profile drug responses across diverse genetic backgrounds

    • Identify patient-specific modulators of GSDMA activity

  • In vivo efficacy validation:

    • Use biotin-conjugated antibodies for pharmacodynamic biomarker development

    • Quantify changes in GSDMA activation in animal tissues after compound treatment

    • Correlate with disease-relevant endpoints

This integration of biotin-conjugated GSDMA antibodies into drug discovery workflows provides opportunities to identify novel therapeutic candidates targeting pyroptotic cell death pathways, with potential applications in inflammatory disorders, infection, and cancer.

What methodological considerations should researchers address when using biotin-conjugated GSDMA antibodies in single-cell analysis techniques?

Single-cell analysis with biotin-conjugated GSDMA antibodies requires specialized methodological considerations to obtain reliable, high-resolution data:

• Single-Cell Protein Analysis Approaches:

  • Mass cytometry (CyTOF) integration:

    • Conjugate GSDMA antibodies with biotin for metal-tagged streptavidin detection

    • Combine with up to 40 additional cellular markers

    • Establish optimal staining concentrations through titration (typically higher dilution than flow cytometry)

    • Implement proper barcoding strategies for batch processing

  • Single-cell Western blotting:

    • Capture single cells in microwell arrays

    • Perform in-situ lysis, protein separation, and immobilization

    • Probe with biotin-conjugated GSDMA antibodies

    • Detect using fluorescent streptavidin conjugates

• Multimodal Single-Cell Analysis:

  • CITE-seq compatibility:

    • Convert biotin-conjugated antibodies to oligonucleotide-tagged versions

    • Simultaneously profile GSDMA protein levels and transcriptome

    • Correlate protein expression with mRNA abundance at single-cell resolution

  • Spatial transcriptomics integration:

    • Use biotin-conjugated GSDMA antibodies in sequential immunofluorescence

    • Overlay with spatial transcriptomics data

    • Create multimodal maps of GSDMA expression and activation states in tissue context

• Technical Optimization for Single-Cell Applications:

  • Signal amplification strategies:

    • Tyramide signal amplification for low-abundance detection

    • Proximity ligation assays for detecting protein-protein interactions

    • Rolling circle amplification for enhanced sensitivity

  • Background reduction approaches:

    • Cell-specific autofluorescence profiling and computational removal

    • Optimized fixation protocols to preserve epitopes while reducing background

    • Careful titration to identify minimal antibody concentration for specific signal

• Analytical Considerations:

  • Data normalization approaches:

    • Use stable housekeeping proteins as internal controls

    • Implement bead-based standards for cross-experiment calibration

    • Apply computational methods to correct batch effects

  • High-dimensional data analysis:

    • Unsupervised clustering to identify cell populations based on GSDMA and other markers

    • Trajectory analysis to map GSDMA activation during cell death processes

    • Visualization techniques (tSNE, UMAP) to represent complex single-cell data

• Validation Framework:

  • Orthogonal validation:

    • Compare single-cell protein measurements with bulk analysis

    • Validate key findings using genetic approaches (CRISPR knockout)

    • Correlate GSDMA activation with functional readouts at single-cell level

  • Technical controls:

    • Include spike-in control cells with known GSDMA expression levels

    • Perform antibody validation on positive and negative control cells

    • Implement isotype controls specific to single-cell platforms

Addressing these methodological considerations enables researchers to generate robust single-cell data on GSDMA expression, localization, and activation states, providing unprecedented insights into the heterogeneity of pyroptotic responses across cell populations.