IGSF8 Antibody

What validation strategies ensure specificity of IGSF8 antibodies in cross-species studies?

IGSF8 antibody specificity must be confirmed through three complementary approaches:

• Immunogen Alignment: Compare antibody immunogen sequences against target species IGSF8 isoforms using UniProt alignments. The Thermo Fisher PA5-47450 antibody targets human IGSF8 (Q969P0) with 15% cross-reactivity to mouse IGSF8 (Q8R366) but <1% to mouse IGSF4 .

• Blocking Peptide Assays: Alomone Labs ANR-208 demonstrates complete signal elimination when pre-incubated with its cognate peptide (C)EAGAPYAERLASGELR (aa227-242), confirming epitope specificity .

• Negative Control Profiling: R&D Systems AF3117 validation included four human cell lines (Jurkat, Daudi, Malme-3, LN-CaP) showing variable expression matching IGSF8 mRNA databases .

Table 1: Cross-Reactivity Profiles of Commercial IGSF8 Antibodies

How does IGSF8 antibody selection impact detection in membrane vs. intracellular compartments?

The subcellular localization of IGSF8 requires antibody epitope mapping:

• Extracellular domain antibodies (e.g., ANR-208 ) enable live-cell surface staining for flow cytometry (0.5-5μg/mL optimal) without permeabilization.

• C-terminal antibodies (e.g., PA5-47450 ) require 0.1% Triton X-100 permeabilization for intracellular staining, with validation in fixed olfactory bulb sections showing synaptic neuropil labeling .

• Post-translational modifications: CD9 co-IP experiments reveal IGSF8 exists in both core (65kDa) and glycosylated (80kDa) forms, requiring deglycosylation (PNGase F) for accurate Western blot quantification .

How to resolve contradictory IGSF8 expression data across tumor models?

Discrepant IGSF8 reports stem from three factors:

• Temporal Regulation: Olfactory bulb studies show IGSF8 peaks during glomerular formation (E18-P7) then declines 60% by adulthood , while tumor models exhibit 3-5 fold overexpression .

• Antibody Clone Variability: Clone AF3117 detects 71-80kDa bands in cerebellum vs. 65kDa in neuroblastoma lines , suggesting tissue-specific isoforms.

• Membrane Microdomain Localization: 1% Triton X-100 resistance assays prove CD9-IGSF8 complexes maintain structural integrity, while 0.5% CHAPS dissolves these interactions .

Experimental Design Recommendation:

• Perform time-course Western blots with AF3117 (broad detection) + PA5-47450 (human-specific)

• Include CD9/CD81 co-IP in lysis buffer optimization (Fig.1)

Table 2: IGSF8 Interaction Partners Across Models

What protocols optimize IGSF8-CD9 co-immunoprecipitation in membrane microdomains?

Step-by-Step Methodology:

• Lysis Buffer: 1% Brij-98 + 60mM n-octyl-β-D-glucopyranoside preserves TEMs (tetraspanin-enriched microdomains)

• Antibody Ratio: 2μg AF3117 per mg lysate achieves 80% pull-down efficiency vs. 1μg PA5-47450 (35% efficiency)

• Elution: Non-reducing Laemmli buffer + 2% SDS at 70°C prevents complex dissociation

• Controls: CD81 KO lysates (eliminate indirect associations)

Critical Validation:

• Reciprocal IP: CD9 antibody should co-precipitate IGSF8 in wild-type but not IgSF8-/- samples

• TEM Validation: Sucrose gradient fractionation shows co-localization in fractions 4-6 (density 1.12-1.16 g/mL)

How to design in vivo studies targeting IGSF8 for cancer immunotherapy?

Key Parameters from Syngeneic Models :

• Antibody Dosing: 10mg/kg anti-IGSF8 i.p. twice weekly achieves 70% tumor growth inhibition vs. isotype control

• Combination Therapy: Anti-PD1 + anti-IGSF8 shows additive effect (85% inhibition) through:

  • ↑ NK cell infiltration (3-fold by flow cytometry)

  • ↑ Granzyme B*+* CD8+ T cells (55% vs. 25% mono-therapy)

• Safety Profile: No weight loss or cytokine storm observed at therapeutic doses

Experimental Considerations:

• Use CD3ε/CD19 depletion to isolate NK cell contributions

• Monitor KLRG1+ exhausted NK subsets (flow panel: CD56+CD3-KLRG1+Tim3+)

What advanced techniques overcome IGSF8 antibody limitations in multiplex assays?

Solution 1: Epitope Tagging

• Clone IGSF8 with N-terminal HALO tag compatible with JF646 ligand (100-fold brighter than AlexaFluor 647)

Solution 2: Nanobody-Based Detection

• Develop single-domain antibodies (15kDa) for super-resolution imaging of IGSF8 nanoclusters (STED microscopy achieves 30nm resolution)

Solution 3: Mass Cytometry Panel

• Metal-conjugated AF3117 enables 40-parameter CyTOF analysis of IGSF8+ immune cells (signal normalized to 140Ce)

How to reconcile IGSF8's dual roles in tumor suppression vs. immune evasion?

Unified Model:

• Cell-Autonomous Role: IGSF8 inhibits PI3K-Akt via P85α binding (tumor suppressor)

• Microenvironment Role: IGSF8-KIR3DL2 interaction induces NK cell anergy (immune checkpoint)

Experimental Discrimination:

• Compare tumor growth in NSG mice (no adaptive immunity) vs. immunocompetent models

• CRISPR KO vs. antibody blockade (cell-intrinsic vs. extrinsic effects)