IGSF3 Antibody, Biotin conjugated

What is IGSF3 and why is it significant in research settings?

IGSF3 (Immunoglobulin Superfamily Member 3) is a 1,194 amino acid protein with a molecular mass of approximately 135.2 kDa that localizes to the cell membrane. It contains an N-terminal signal peptide, eight immunoglobulin (Ig) domains, and a transmembrane segment, suggesting its function as a surface receptor or cell adhesion molecule . IGSF3 is widely expressed, with predominant expression in kidney, placenta, and lung tissues .

Recent research has identified IGSF3 as particularly significant due to its association with severe COPD (Chronic Obstructive Pulmonary Disease), where a loss of IGSF3 expression due to germline mutation was identified in a patient with severe emphysema . Additionally, IGSF3 has been linked to lacrimal duct defects, making it an important target for researchers investigating respiratory and ophthalmological conditions .

What are the key specifications of commercially available IGSF3 Antibody, Biotin conjugated?

The biotin-conjugated IGSF3 polyclonal antibody is typically derived from rabbit hosts, using KLH-conjugated synthetic peptides from human IGSF3 (commonly spanning amino acids 631-730/1194) as immunogens . This antibody preparation is generally:

• Purified using Protein A

• Concentrated at approximately 1μg/μl

• Stored in aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol

• Recommended for storage at -20°C for approximately 12 months

The biotin conjugation enables versatile detection methods, particularly in ELISA applications, offering enhanced sensitivity through streptavidin-based detection systems .

What experimental applications are optimized for IGSF3 Antibody, Biotin conjugated?

While non-conjugated IGSF3 antibodies are commonly used in Western Blot (WB), Immunohistochemistry (IHC-P), and Immunofluorescence (IF), the biotin-conjugated version has specific advantages in certain applications:

The biotin conjugation is particularly valuable for multiplex assays where researchers need to detect multiple targets simultaneously using different visualization systems .

How should researchers design experiments to validate IGSF3 antibody specificity in their experimental system?

A comprehensive validation strategy should include:

• Positive and negative control tissues/cells: Use tissues known to express IGSF3 highly (kidney, placenta, lung) as positive controls and tissues with minimal expression as negative controls .

• Knockdown/knockout validation: Compare staining between wildtype samples and those where IGSF3 expression has been reduced (siRNA) or eliminated (CRISPR-Cas9).

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to verify specific binding is blocked.

• Cross-reactivity testing: Test the antibody against recombinant IGSF3 proteins from different species (human, mouse, rat) to confirm the species reactivity claims .

• Multi-method confirmation: Compare results using alternative detection methods (e.g., mass spectrometry) to confirm target identity and size.

This methodical approach ensures that experimental results genuinely reflect IGSF3 biology rather than non-specific binding interactions.

What are the optimal fixation and antigen retrieval protocols for IGSF3 detection in tissue sections?

Based on experimental evidence with IGSF3 antibodies:

Research studying IGSF3 in mouse lungs exposed to cigarette smoke successfully used these protocols to demonstrate decreased IGSF3 expression in both lung parenchyma and airway epithelium after prolonged smoke exposure .

How can researchers optimize IGSF3 antibody detection in co-localization studies with tetraspanin markers?

IGSF3 has been demonstrated to interact with tetraspanins (CD9, CD81, CD82) and integrin β1 within tetraspanin-enriched microdomains (TEMs) . For optimal co-localization studies:

• Sample preparation: Use mild detergents (e.g., 1% Brij 98 or CHAPS) that preserve tetraspanin-enriched microdomain integrity.

• OptiPrep density gradient separation: Isolate TEMs using 5-40% OptiPrep density gradient ultracentrifugation, collecting fractions for analysis. Tetraspanins and IGSF3 typically co-localize in fractions 8-12 .

• Co-immunoprecipitation: Use anti-CD9 antibody for immunoprecipitation followed by IGSF3 immunoblotting to confirm direct interaction.

• Multiplexed imaging: For immunofluorescence co-localization, use spectrally distinct fluorophores (primary antibody combinations should be from different host species).

• Control for antibody cross-reactivity: Include single-stained controls to establish specificity when using multiple primary antibodies.

This approach successfully demonstrated that IGSF3 co-resides with tetraspanins and integrin β1 in cell membranes and specifically interacts with CD9, even after cigarette smoke extract exposure .

What strategies help overcome weak or inconsistent signal when using biotin-conjugated IGSF3 antibodies?

When experiencing weak signals with biotin-conjugated IGSF3 antibodies, consider these methodological adjustments:

• Optimize antibody concentration: Test a dilution series (e.g., 1:100, 1:200, 1:400, 1:800) to identify the optimal working dilution for your specific sample.

• Enhance antigen retrieval: Extended HIER times (up to 30 minutes) or testing alternative buffers (EDTA pH 8.0 vs. citrate pH 6.0) may improve epitope accessibility.

• Block endogenous biotin: Particularly in biotin-rich tissues (kidney, liver), use commercial avidin/biotin blocking kits before antibody application.

• Extend incubation time: Overnight incubation at 4°C often yields stronger specific signals than shorter incubations at room temperature.

• Signal amplification systems: Implement tyramide signal amplification (TSA) or catalyzed reporter deposition methods to enhance detection sensitivity.

• Fresh antibody aliquots: Avoid repeated freeze-thaw cycles by preparing single-use aliquots; storage conditions significantly impact antibody performance .

These approaches have successfully optimized IGSF3 detection in cases where standard protocols yielded insufficient signal intensity.

How can researchers address high background when using biotin-conjugated IGSF3 antibodies in tissues with endogenous biotin?

High background is particularly challenging with biotin-conjugated antibodies due to endogenous biotin in many tissues. Effective mitigation strategies include:

• Avidin/biotin blocking: Apply an avidin/biotin blocking kit sequentially before antibody incubation to saturate endogenous biotin sites.

• Alternative detection systems: Consider switching to non-biotin detection methods if background persists despite blocking.

• Extended washing steps: Implement additional washing steps with PBS-T (0.1% Tween-20) to reduce non-specific binding.

• Optimize blocking buffers: Test different blocking solutions (5% BSA, 5% normal serum, commercial blockers) to identify optimal background reduction.

• Reduce primary antibody concentration: A more dilute antibody solution may reduce non-specific binding while maintaining specific signal.

• Pre-absorption: Incubate the antibody with liver powder (high in endogenous biotin) to pre-absorb components causing non-specific binding.

Researchers studying IGSF3 in lung tissues have successfully implemented these approaches to distinguish specific signals from background .

What are potential sources of cross-reactivity with IGSF3 antibodies and how can they be mitigated?

IGSF3 shares structural similarities with other immunoglobulin superfamily members, potentially leading to cross-reactivity. Common cross-reactivity issues and mitigation strategies include:

Additionally, proper antibody validation using genetic manipulations (siRNA knockdown, CRISPR knockout) provides the most definitive confirmation of specificity. When using patient-derived samples, comparing results from multiple antibodies targeting different IGSF3 epitopes can help confirm specificity .

How can IGSF3 antibody biotin conjugates be employed in investigating IGSF3's role in COPD pathogenesis?

Recent research identified a loss of IGSF3 expression due to germline mutation in a patient with severe emphysema, establishing a potential causal link between IGSF3 deficiency and COPD development . To further investigate this relationship, researchers can employ biotin-conjugated IGSF3 antibodies in several sophisticated approaches:

• Patient stratification: Screen COPD patient cohorts using IHC to correlate IGSF3 expression levels with disease severity, progression, and specific phenotypes.

• Smoke exposure models: Quantify IGSF3 expression changes in response to cigarette smoke using animal models. Research has already demonstrated that both acute (1 day) and chronic (4 months) cigarette smoke exposure significantly reduces IGSF3 expression in mouse lungs .

• Pathway analysis: Combine IGSF3 detection with markers of unfolded protein response and ceramide pathways, which show alterations in IGSF3-deficient cells.

• TEM integrity assessment: Investigate whether IGSF3 deficiency disrupts tetraspanin-enriched microdomain organization using co-immunoprecipitation and density gradient fractionation.

• Genetic association studies: Correlate IGSF3 SNPs (particularly rs1414272, rs12066192, and rs6703791) with COPD severity and exacerbation frequency .

This multifaceted approach can provide deeper insights into how IGSF3 dysfunction contributes to COPD pathogenesis at both cellular and clinical levels.

What methodological approaches enable effective investigation of IGSF3's interaction with the tetraspanin network?

IGSF3 has been confirmed as a tetraspanin-interacting protein that colocalizes with CD9 and integrin β1 in tetraspanin-enriched domains . To thoroughly investigate these interactions:

• Density gradient fractionation: Use OptiPrep density gradient ultracentrifugation (5-40%) to isolate TEMs. IGSF3 consistently appears in fractions 8-12 along with tetraspanins CD82, CD9, CD81, and TSPAN7 .

• Co-immunoprecipitation analysis: Employ anti-CD9 antibody for immunoprecipitation followed by IGSF3 immunoblotting to confirm direct binding interactions. This approach successfully demonstrated that cigarette smoke does not disrupt the CD9-IGSF3 interaction .

• Surface biotinylation: Use cell surface biotinylation followed by streptavidin pulldown and IGSF3 immunoblotting to confirm IGSF3's presence on the cell surface alongside other TEM components .

• Subcellular fractionation: Combine with organelle-specific markers (PDI for endoplasmic reticulum, CytC and SDHA for mitochondria, LC3B for autophagosomes) to map IGSF3's intracellular distribution .

• Super-resolution microscopy: Apply techniques like STORM or PALM with dual-labeled antibodies to visualize IGSF3-tetraspanin interactions at nanoscale resolution.

These complementary approaches provide a comprehensive view of IGSF3's role within the tetraspanin network under both normal and pathological conditions.

How can researchers effectively compare results between different IGSF3 antibodies in experimental systems?

• Epitope mapping: Document the specific IGSF3 regions targeted by each antibody. The commonly used rabbit polyclonal targets amino acids 631-730/1194 , but antibodies targeting different epitopes may yield varying results.

• Cross-validation matrix: Create a validation matrix where each antibody is tested across multiple techniques (WB, IHC, IF, IP) with standardized positive and negative controls.

• Isoform specificity assessment: Determine which antibodies detect both IGSF3 isoforms versus those that are isoform-specific. The two IGSF3 isoforms differ by an additional 60bp exon in isoform 1 .

• Quantitative comparison: For quantitative applications, establish standard curves using recombinant IGSF3 and determine the linear detection range for each antibody.

• Batch consistency verification: When obtaining new antibody lots, perform side-by-side comparisons with previous lots on identical samples to ensure consistent performance.

• Conjugation effects assessment: Directly compare unconjugated versus biotin-conjugated antibodies to determine if conjugation affects epitope recognition or binding affinity.

This systematic approach ensures that experimental outcomes reflect true biological phenomena rather than variations in antibody characteristics.

How should researchers interpret changes in IGSF3 expression patterns in smoking-related lung pathologies?

Research has demonstrated that cigarette smoke exposure significantly reduces IGSF3 expression in mouse lungs, both after acute (1 day) and chronic (4 months) exposure . When interpreting IGSF3 expression patterns:

• Cellular specificity: Analyze expression changes in specific cell types separately. IGSF3 shows differential expression between lung parenchyma and airway epithelium, with both showing marked reduction after smoke exposure .

• Temporal dynamics: Distinguish between acute responses (potentially protective/adaptive) and chronic changes (potentially pathological). The rapid decrease after just one day of smoke exposure suggests IGSF3 downregulation may be an early event in smoke-induced pathology .

• Correlation with clinical parameters: For human samples, correlate IGSF3 expression levels with lung function parameters (FEV1, FVC), disease severity indices, and exacerbation frequency.

• Genetic context: Consider IGSF3 SNP status when interpreting expression data. Specific SNPs (rs1414272, rs12066192) have been directly associated with COPD severity, while others (rs6703791) show inverse association .

• Pathway integration: Interpret IGSF3 changes in context with alterations in unfolded protein response and ceramide pathways, which show significant alterations in IGSF3-deficient cells .

This contextual interpretation provides deeper insights into IGSF3's role in normal lung homeostasis and disease pathogenesis.

What quantitative methods provide the most reliable analysis of IGSF3 expression in complex tissue samples?

For accurate quantification of IGSF3 in heterogeneous tissues:

• Digital pathology approaches: Use automated image analysis software with tissue segmentation to quantify IGSF3 immunostaining in specific cell types within complex tissues.

• Multiplex immunofluorescence: Combine IGSF3 staining with cell-type-specific markers to quantify expression in distinct cell populations simultaneously.

• Laser capture microdissection: Isolate specific cell populations before protein/RNA extraction to obtain cell-type-specific quantification.

• Single-cell proteomics/transcriptomics: Apply emerging single-cell technologies to measure IGSF3 expression at individual cell resolution, revealing population heterogeneity.

• Reference gene/protein normalization: Use multiple housekeeping controls appropriate for the specific tissue type to ensure reliable normalization.

• Standard curve calibration: Include recombinant IGSF3 standards or calibrated tissue microarrays as internal controls across experiments.

These methodologies have demonstrated superior reliability when quantifying membrane proteins like IGSF3 in heterogeneous samples such as lung tissue, where cellular composition varies considerably between normal and diseased states .

How can researchers distinguish between IGSF3 expression changes and alterations in protein localization?

Changes in IGSF3 staining patterns may reflect altered expression, localization, or both. To differentiate:

• Subcellular fractionation: Separate membrane, cytosolic, and organelle fractions to determine if total IGSF3 levels change or if just the distribution shifts between compartments.

• Co-localization analysis: Quantify co-localization coefficients with markers for different cellular compartments (plasma membrane, endoplasmic reticulum, endosomes) under normal and experimental conditions.

• Surface biotinylation: Compare surface-exposed IGSF3 (detected by biotinylation followed by streptavidin pulldown) with total cellular IGSF3 to determine if membrane trafficking is affected .

• Pulse-chase experiments: Use inducible tagged IGSF3 constructs to track protein movement through cellular compartments over time.

• Native versus denatured detection: Compare native condition immunoprecipitation (reflecting accessible epitopes) with denatured western blotting (reflecting total protein) to identify conformational changes or masking.

Research has shown that cigarette smoke exposure does not alter IGSF3's presence at the plasma membrane despite reducing total expression, indicating that reduced synthesis rather than altered trafficking may be the primary mechanism .

How can biotin-conjugated IGSF3 antibodies facilitate research into IGSF3's potential role in autophagy?

IGSF3 has been detected in LC3B-positive fractions (10 and 16) during cellular fractionation studies, suggesting potential localization in autophagosome membranes . To investigate this emerging connection:

• Co-localization studies: Use biotin-conjugated IGSF3 antibodies with fluorescent streptavidin in combination with autophagy markers (LC3B, p62) to visualize potential co-localization in autophagosomal structures.

• Autophagy modulation: Examine how IGSF3 expression/localization changes during induced autophagy (starvation, rapamycin) or autophagy inhibition (bafilomycin A1, chloroquine).

• IGSF3 knockout impact: Analyze how IGSF3 deletion affects autophagic flux using LC3B-II/I ratio analysis and p62 accumulation assays.

• Selective autophagy assessment: Investigate whether IGSF3 is involved in selective autophagy pathways by examining its interaction with specific cargo receptors.

• Membrane dynamics: Use live-cell imaging with fluorescently tagged IGSF3 to track its movement during autophagosome formation and maturation.

This research direction could reveal previously uncharacterized functions of IGSF3 in cellular quality control mechanisms, potentially connecting to the unfolded protein response alterations observed in IGSF3-deficient cells .

What methodological approaches can best investigate IGSF3's potential roles in unfolded protein response and ceramide pathways?

IGSF3-deficient patient-derived lymphoblastoids exhibit multiple alterations in gene expression, especially in the unfolded protein response and ceramide pathways . To investigate these connections:

• ER stress induction: Compare UPR marker expression (BiP/GRP78, CHOP, XBP1 splicing) in IGSF3-normal versus IGSF3-deficient cells under basal and ER stress conditions (tunicamycin, thapsigargin).

• Lipidomic analysis: Perform comprehensive lipidomic profiling focused on ceramide species and related sphingolipids in IGSF3-manipulated cells.

• Pathway inhibition studies: Use specific inhibitors of ceramide synthesis (myriocin, fumonisin B1) or ER stress (TUDCA, 4-PBA) to determine if they rescue phenotypes associated with IGSF3 deficiency.

• Protein-lipid interaction assays: Investigate direct interaction between IGSF3 and ceramides using protein-lipid overlay assays or liposome binding experiments.

• Integrated transcriptomics: Perform RNA-seq on IGSF3-normal and deficient cells under various stress conditions, focusing on UPR and lipid metabolism gene networks.

• In situ proximity ligation: Detect potential interactions between IGSF3 and key UPR sensors (IRE1α, PERK, ATF6) or ceramide metabolism enzymes.

These approaches could uncover mechanistic links between IGSF3, cellular stress responses, and membrane lipid composition that underlie its contribution to disease states .