Slfn9 Antibody, HRP conjugated

What is Slfn9 and what biological roles does it serve?

Slfn9 (Schlafen family member 9) is an endoribonuclease that cleaves tRNAs and rRNAs . Recent research has established Slfn9 as an innate immune sensor for intracellular single-stranded DNA (ssDNA), particularly those containing CGT motifs . In mice, Slfn9 functions as the homologue of human SLFN11, with both proteins playing critical roles in immune response pathways . The Slfn9 protein contributes to cytokine expression and cell death mechanisms following detection of pathogen-derived or endogenous ssDNA. This understanding is significant as it establishes CGT ssDNA and SLFN9 as a novel type of immunostimulatory nucleic acid and pattern recognition receptor, respectively .

How does mouse Slfn9 relate to human SLFN11 in research contexts?

Mouse Slfn9 shares orthologous functions with human SLFN11 despite the rapid diversification of the SLFN family members . Research demonstrates that:

• Both mSLFN8/9 and hSLFN11 are recruited rapidly to microlaser-irradiated DNA damage tracks

• Slfn8/9 expression can complement SLFN11 loss in human SLFN11−/− cells

• Expression of either gene reduces growth rate to wild-type levels

• Both partially restore sensitivity to DNA-damaging agents

• Both accelerate stalled fork degradation and decrease RPA and RAD51 foci numbers after DNA damage

These functional similarities make mouse models viable for studying the biological roles of SLFN11, facilitating in vivo studies that might inform human disease understanding and therapeutic development .

What are the optimal applications and conditions for Slfn9 Antibody, HRP conjugated?

Based on validated protocols, the Slfn9 Antibody, HRP conjugated performs optimally in:

Storage conditions significantly impact antibody performance:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles

• Buffer composition (50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative) maintains stability

When conducting experiments, proper controls should include:

• Positive control: Mouse spleen or activated immune cells

• Negative control: Samples from Slfn9 knockout mice (when available)

• Isotype control: Rabbit IgG-HRP at equivalent concentration

How should researchers troubleshoot when Slfn9 antibody shows unexpected results in immune cell studies?

When faced with unexpected results using Slfn9 antibody in immune cell studies, implement this systematic troubleshooting approach:

How can Slfn9 antibody be utilized to study the protein's role in ssDNA sensing pathways?

To investigate Slfn9's role in ssDNA sensing pathways:

• Co-localization studies: Use Slfn9 antibody in immunofluorescence together with fluorescently labeled CGT-motif ssDNA to visualize binding interactions. Research has shown that CGT ODNs trigger cytosolic translocation of SLFN proteins and colocalize with cytosolic SLFN11 in human cells, with SLFN9 showing similar behavior in mouse cells .

• Immunoprecipitation approaches:

  • Perform chromatin immunoprecipitation (ChIP) using Slfn9 antibody to identify DNA binding sites

  • Couple with mass spectrometry to identify protein complexes formed during ssDNA sensing

• Functional response measurement:

  • Monitor changes in Slfn9 localization after ssDNA transfection

  • Quantify downstream cytokine expression (particularly IFNβ1, IL6, and CXCL2) in wild-type versus Slfn9-deficient cells

  • Compare responses to different ssDNA motifs (CGT motifs versus other sequences)

• Mechanistic analysis:

  • Study differential responses between stimulatory CGT ODNs and canonical TLR9-sensed CpG ODNs

  • Evaluate methylation tolerance of Slfn9 binding to ssDNA

What is the significance of studying Slfn9 in cancer immunology research, and how can the antibody facilitate this work?

The Slfn9 antibody is invaluable for cancer immunology research based on several key findings:

• Tumor immune infiltration correlation: SLFN family members show significant correlations with tumor-infiltrating immune cells (TIICs), particularly:

  • CD8+ T cells

  • Dendritic cells

  • Macrophages

• Cancer prognostic potential: SLFN family proteins may serve as prognostic indicators in multiple cancers:

  • Gastric cancer shows associations between SLFN expression and immune checkpoint expression

  • SLFN11 (human homologue of mouse Slfn9) influences macrophage movement and M2-like differentiation in hepatocellular carcinoma, affecting PD-L1 expression through NF-κB signaling

• Therapeutic response prediction:

  • SLFN11 status predicts response to DNA-damaging agents in cancer therapy

  • SLFN9, as a functional orthologue, may similarly impact therapeutic sensitivity

• Research methodology using the antibody:

  • Use in immunohistochemistry to quantify Slfn9 expression in tumor sections

  • Employ for flow cytometry to analyze Slfn9 in tumor-infiltrating immune cells

  • Combine with immune cell markers to study correlations between Slfn9 expression and specific immune populations

How should researchers interpret differences in Slfn9 detection between mouse and human samples?

When analyzing cross-species differences in Slfn9/SLFN11 detection:

• Evolutionary context: The SLFN family has undergone rapid diversification, resulting in species-specific patterns:

  • Mice lack the SLFN11 gene found in humans

  • Mouse Slfn9 functionally complements human SLFN11

• Functional equivalence assessment:

  • Evaluate both proteins for DNA damage recruitment kinetics

  • Test complementation of SLFN11 deficiency with mouse Slfn9 expression

  • Compare sensitivity to DNA-damaging agents

• Expression pattern differences:

  • Human SLFN11 expression varies across cancer types

  • Mouse Slfn9 expression may show tissue-specific patterns

• Experimental design considerations:

  • Use species-specific antibodies (anti-mouse Slfn9 vs. anti-human SLFN11)

  • Include appropriate positive controls for each species

  • Consider developing targeted assays for orthologous functions rather than relying solely on protein detection

What methodological approaches can resolve contradictory data when studying Slfn9's role in immune responses?

When facing contradictory data regarding Slfn9's immune functions:

• Cell type specificity analysis:

  • Compare Slfn9 responses across different immune cell populations

  • Assess expression and function in primary bone marrow-derived macrophages versus plasmacytoid dendritic cells and other immune cells

  • Evaluate tissue-resident versus circulating immune cells

• Stimulation specificity verification:

  • Test differential responses to various stimuli:

    • CGT-containing ssDNA (responsive to Slfn9)

    • Poly(dA:dT), poly(I:C), or LPS (Slfn9-independent pathways)

    • Different types of CpG ODNs (ODN1585, ODN1826, ODN2395)

• Genetic validation approaches:

  • Utilize Slfn9 knockout models to confirm specificity

  • Employ gene silencing with targeted siRNAs

  • Use complementation experiments with wild-type and mutant Slfn9

• Technical consideration matrix:

How might Slfn9 antibodies contribute to developing novel immunotherapeutic approaches?

The Slfn9 antibody can advance immunotherapeutic research through:

• Biomarker development:

  • SLFN11 shows potential as a biomarker for predicting response to immune checkpoint inhibitors in advanced liver cancer

  • Slfn9, as a functional homologue, could serve as a parallel biomarker in mouse models

• Therapeutic target identification:

  • Mapping Slfn9-dependent pathways may reveal novel targets

  • Understanding how Slfn9 modulates immune responses could inform combination therapies

• Resistance mechanism studies:

  • Investigating how Slfn9 expression affects tumor response to immunotherapy

  • Exploring connections between Slfn9, macrophage polarization, and PD-L1 expression

• Translation to clinical applications:

  • Developing companion diagnostics for immunotherapies

  • Creating screening tools to identify patients likely to respond to specific treatments

What experimental strategies can best elucidate the structural-functional relationship of Slfn9 using antibody-based approaches?

To investigate Slfn9's structure-function relationships:

• Domain-specific antibody application:

  • Use antibodies targeting different domains (the antibody in question targets AA 1-83)

  • Compare binding profiles across functional domains:

    • N-terminal AAA_4 domain

    • SLFN box

    • SWAVDL domain (in subgroup II/III members)

    • DNA/RNA helicase domain (C-terminal, subgroup III only)

• Mutation-function correlation studies:

  • Express mutant Slfn9 proteins with domain deletions or point mutations

  • Use antibodies to assess expression, stability, and localization

  • Correlate structural alterations with functional outcomes

• Interaction network mapping:

  • Employ co-immunoprecipitation with Slfn9 antibody to identify binding partners

  • Use proximity labeling techniques coupled with antibody pulldown

  • Analyze how different structural elements contribute to protein-protein interactions

• Dynamic structural changes visualization:

  • Study conformational changes upon ssDNA binding

  • Monitor translocation between nucleus and cytoplasm during immune activation

  • Assess oligomerization state under different cellular conditions