SLAMF6 Antibody, Biotin conjugated

What is SLAMF6 and what are its major biological functions?

SLAMF6 is a homotypic receptor of the immunoglobulin superfamily constitutively expressed on T cells, NK cells, B cells, and dendritic cells. Its primary functions involve immune cell signaling and modulation. Research indicates SLAMF6 plays a complex role in T cell responses, with evidence suggesting it acts as an inhibitory checkpoint receptor when expressed on T cells targeting cancer cells. SLAMF6 enhances T cell function by increasing cellular adhesiveness through activation of the small GTPase Rap1 . Interestingly, SLAMF6 deficiency has been shown to augment tumor-killing capacity and skew toward enhanced anti-tumor immunity in experimental models .

What distinguishes biotin-conjugated SLAMF6 antibodies from non-conjugated versions?

Biotin-conjugated SLAMF6 antibodies contain covalently attached biotin molecules that enable high-affinity binding to streptavidin and avidin. This conjugation offers significant advantages in detection sensitivity and versatility compared to non-conjugated antibodies. The biotin-streptavidin interaction (Kd = 10^-15 M) is among the strongest non-covalent biological interactions known, providing excellent signal amplification in various assay formats. While standard SLAMF6 antibodies like catalog #A05310 are suitable for direct detection in Western blot and ELISA applications , the biotin-conjugated versions offer enhanced detection sensitivity and compatibility with multiple visualization systems through secondary detection with streptavidin-conjugated reporters .

What is the molecular structure and weight of SLAMF6 and how does this impact antibody binding?

The calculated molecular weight of SLAMF6 is approximately 37 kDa, yet the observed weight in experimental conditions is typically around 111 kDa . This significant discrepancy is attributable to post-translational modifications, particularly glycosylation of the extracellular domain. The SLAMF6 protein structure features an extracellular region with immunoglobulin-like domains, a transmembrane segment, and cytoplasmic tail containing immunoreceptor tyrosine-based switch motifs (ITSMs). These ITSMs are crucial for downstream signaling and interaction with adaptor proteins including SAP (SLAM-Associated Protein). Antibodies raised against different epitopes, particularly those in the 20-100 or 248-331 amino acid ranges, may exhibit different binding characteristics and functional effects when used in experimental systems .

What are the optimal protocols for using biotin-conjugated SLAMF6 antibodies in ELISA assays?

For optimal use of biotin-conjugated SLAMF6 antibodies in ELISA, follow this methodological approach:

• Preparation: Briefly spin down the biotin conjugate before use. Add 100 μL of 1X Assay Diluent to prepare a biotin conjugate concentrate .

• Dilution optimization: Perform titration experiments to determine optimal working concentration. For biotin-conjugated SLAMF6 antibodies, recommended starting dilutions are typically 1:5000-1:20000 for ELISA applications .

• Incubation parameters: After coating plates with capture antibody and blocking, add samples followed by the biotin-conjugated SLAMF6 antibody. Incubate at room temperature (20-25°C) for 1-2 hours with gentle shaking.

• Detection system: Use streptavidin-HRP (recommended dilution 1:200) followed by TMB substrate for colorimetric detection .

• Quality controls: Include appropriate negative controls (isotype-matched irrelevant antibodies) and positive controls (recombinant SLAMF6 protein) in each assay.

For sandwich ELISA specifically, ensure the biotin-conjugated detection antibody recognizes a different epitope than the capture antibody to avoid steric hindrance issues.

How should researchers optimize Western blot protocols using biotin-conjugated SLAMF6 antibodies?

Optimizing Western blot protocols with biotin-conjugated SLAMF6 antibodies requires attention to several key methodological aspects:

• Sample preparation: Create cell lysates using RIPA buffer and separate proteins by Tris-glycine PAGE before semi-dry transfer to nitrocellulose membranes .

• Blocking and antibody dilution: Use 3-5% BSA in TBST for blocking and antibody dilution to minimize background. For biotin-conjugated SLAMF6 antibodies, begin with dilutions of 1:500-1:2000 .

• Detection strategy: Utilize streptavidin conjugated to HRP, IR dye, or fluorescent reporter for visualization. For infrared imaging systems like Licor CLX, IRDye 680RD or 800CW-conjugated streptavidin provides excellent sensitivity and quantitative capacity .

• Expected molecular weight: Anticipate detection around 111 kDa despite the calculated molecular weight of 37 kDa, due to post-translational modifications .

• Controls and validation: Include positive controls (recombinant SLAMF6) and negative controls (lysates from SLAMF6-negative cells) to validate specificity and minimize misinterpretation of non-specific bands.

When troubleshooting, consider that bands outside expected regions are typically non-specific and may appear randomly when conditions are repeated independently .

What are the storage and handling requirements to maintain optimal activity of biotin-conjugated SLAMF6 antibodies?

Proper storage and handling are critical for maintaining the functional integrity of biotin-conjugated SLAMF6 antibodies:

• Long-term storage: Store at -20°C for up to one year or at -80°C for extended periods. Biotin-conjugated antibodies are typically supplied in stabilizing buffers containing 50% glycerol and 0.01M PBS at pH 7.4, often with preservatives like 0.03% Proclin 300 .

• Working storage: For frequent use over short periods (up to one month), store at 4°C to avoid repeated freeze-thaw cycles .

• Aliquoting strategy: Upon receipt, divide into small single-use aliquots before freezing to minimize freeze-thaw cycles, which can cause biotin dissociation and protein denaturation.

• Thawing procedure: Thaw aliquots completely at room temperature or 4°C, followed by gentle mixing without vortexing to prevent protein denaturation.

• Centrifugation step: Briefly spin down the biotin conjugate before each use to collect reagent at the bottom of the tube and ensure accurate pipetting .

Following these guidelines will help maintain antibody specificity and sensitivity across experimental applications.

How can biotin-conjugated SLAMF6 antibodies be utilized to investigate T cell-mediated anti-tumor responses?

Biotin-conjugated SLAMF6 antibodies offer sophisticated approaches for investigating T cell anti-tumor responses:

• Flow cytometry-based functional assays: Use biotin-conjugated anti-SLAMF6 with streptavidin-fluorophores to quantify SLAMF6 expression levels on tumor-infiltrating lymphocytes versus peripheral T cells. This approach enables correlation between SLAMF6 expression and cytotoxic function through multi-parameter analysis of activation markers, exhaustion markers, and cytokine production.

• Mechanistic studies: Research has demonstrated that SLAMF6 deficiency augments tumor killing capacity of CD8+ T cells against melanoma. Biotin-conjugated antibodies can be used to block SLAMF6 in vitro, mimicking genetic deficiency to assess impact on cytokine production, cytotoxicity, and activation of signaling pathways like ERK, ZAP70, and AKT phosphorylation .

• Immunoprecipitation and co-immunoprecipitation: Utilize biotin-conjugated SLAMF6 antibodies with streptavidin beads to pull down SLAMF6 and associated proteins, revealing interaction partners involved in immune synapse formation and signal transduction.

• Imaging studies: Employ biotin-conjugated antibodies with quantum dot-streptavidin conjugates for high-resolution imaging of immune synapses between T cells and tumor cells, enabling quantification of SLAMF6 recruitment to the synapse during tumor cell engagement.

These techniques have revealed that SLAMF6 acts as an inhibitory checkpoint receptor, with SLAMF6-deficient CD8+ T cells displaying enhanced anti-melanoma activity and more effectively preventing melanoma growth compared to T cells with intact SLAMF6 .

What clustering mechanisms affect biotin-conjugated SLAMF6 antibody efficacy in T cell activation studies?

The efficacy of biotin-conjugated SLAMF6 antibodies in T cell activation studies is significantly influenced by clustering mechanisms:

• Spatial organization requirements: Research demonstrates that SLAMF6 clustering is required to augment T cell activation . Biotin-conjugated antibodies offer an advantage in studying this phenomenon as multiple biotin molecules per antibody allow cross-linking through streptavidin, creating controlled oligomerization.

• Soluble versus immobilized antibody effects: Experimental data show differential T cell responses when stimulated with:

  • Soluble antibodies: anti-CD3/anti-mouse IgG (3.25 μg/mL) with anti-SLAMF6 (3.25 μg/mL)

  • Immobilized antibodies: anti-CD3/anti-mouse IgG (1.5 μg/mL) with anti-SLAMF6 (5 μg/mL)

  Immobilization creates antibody clusters that more effectively mimic physiological receptor engagement.

• Whole antibody versus Fab fragment comparison: Full biotin-conjugated SLAMF6 antibodies induce clustering through bivalent binding and potential cross-linking, while monovalent Fab fragments (3.25 μg/mL with anti-mouse IgG at 1.63 μg/mL) fail to induce effective clustering . This methodological distinction is crucial when designing experiments to distinguish between clustering-dependent and independent effects.

• Downstream signaling effects: SLAMF6 clustering enhances T cell function by increasing adhesiveness through activation of the small GTPase Rap1 , which can be monitored using activation assays following stimulation with clustered biotin-conjugated antibodies.

These clustering-dependent mechanisms highlight the importance of antibody presentation method when designing experiments to study SLAMF6 function in T cell biology.

How can researchers use biotin-conjugated SLAMF6 antibodies to investigate its role as an immune checkpoint molecule?

Investigating SLAMF6 as an immune checkpoint molecule requires sophisticated experimental approaches enabled by biotin-conjugated antibodies:

• Checkpoint blockade models: Biotin-conjugated SLAMF6 antibodies can be used to block SLAMF6 homotypic interactions in vitro and in vivo. Research has shown that SLAMF6 deficiency in CD8+ T cells improves anti-melanoma activity, suggesting SLAMF6 acts as an inhibitory checkpoint receptor . In experimental designs:

  • Use Fab fragments to block without inducing clustering

  • Employ whole antibodies to potentially induce signaling while blocking

• Trans-interaction studies: Experimental evidence demonstrates that SLAMF6 expressed in trans by melanoma targets inhibits antitumor T cell reactivity . Biotin-conjugated antibodies can be used to:

  • Block these trans-interactions

  • Quantify binding kinetics between T cell and tumor cell SLAMF6 proteins

  • Visualize molecular distribution at the immune synapse

• Combinatorial checkpoint investigation: Studies combining SLAMF6 blockade with established checkpoint inhibitors (anti-PD-1, anti-CTLA-4) can utilize biotin-conjugated antibodies for:

  • Flow cytometry to measure expression correlation

  • Pull-down assays to identify shared signaling nodes

  • In vivo tracking of multiple checkpoint receptors simultaneously

• SAP-dependent signaling analysis: Since SLAMF6 inhibitory function appears more pronounced in the absence of SAP (SLAM-Associated Protein) , biotin-conjugated antibodies facilitate:

  • Co-immunoprecipitation studies of SLAMF6-SAP complexes

  • Comparative signaling studies in SAP-sufficient and SAP-deficient models

  • Analysis of SLAMF6 binding to alternative partners like PTPN6/SHP-1 via ITSMs

These approaches enable comprehensive investigation of SLAMF6's role in regulating immune responses to tumors and potential therapeutic applications.

How should researchers address epitope masking issues when using biotin-conjugated SLAMF6 antibodies?

Epitope masking is a significant challenge when using biotin-conjugated SLAMF6 antibodies, particularly in complex samples or multi-step protocols. Researchers should employ these methodological approaches:

• Epitope mapping and selection: Different antibodies target distinct regions of SLAMF6, such as amino acids 20-100 or 248-331 . When designing multi-antibody experiments:

  • Use antibodies targeting non-overlapping epitopes

  • Consider the accessibility of epitopes in native versus denatured conditions

  • Account for potential steric hindrance from biotin moieties

• Optimizing denaturation and retrieval protocols:

  • For fixed tissues or cells: Test multiple antigen retrieval methods (heat-induced in citrate buffer pH 6.0 versus EDTA buffer pH 9.0)

  • For Western blotting: Compare reducing versus non-reducing conditions to determine optimal epitope exposure

• Sequential antibody application:

  • In co-staining experiments, apply antibodies recognizing potentially competing epitopes sequentially rather than simultaneously

  • Include washing steps between applications to remove unbound antibodies

• Validation approaches:

  • Confirm staining patterns using multiple antibodies against different SLAMF6 epitopes

  • Include SLAMF6-deficient samples as negative controls to verify specificity

  • Compare biotin-conjugated versus unconjugated antibody performance to identify conjugation-specific issues

• Technical controls for biotin interference:

  • Block endogenous biotin with avidin/streptavidin before applying biotin-conjugated antibodies

  • Include biotin-conjugated isotype controls at matching concentrations

These methodological adjustments help ensure accurate detection and minimize false-negative results due to epitope masking issues.

What are the common pitfalls in data interpretation when using biotin-conjugated SLAMF6 antibodies?

Several common pitfalls affect data interpretation when using biotin-conjugated SLAMF6 antibodies:

• Molecular weight discrepancies: The observed molecular weight of SLAMF6 (111 kDa) significantly differs from the calculated weight (37 kDa) . Researchers should:

  • Anticipate this discrepancy in Western blot analysis

  • Consider running deglycosylation controls to confirm band identity

  • Use positive controls (recombinant SLAMF6) to validate band patterns

• Non-specific binding artifacts: Non-specific bands may appear randomly when conditions are repeated independently . To address this:

  • Always include isotype controls at matching concentrations

  • Perform blocking titration experiments to determine optimal conditions

  • Consider pre-adsorption of antibodies with recombinant protein to confirm specificity

• Signal interference from endogenous biotin: Endogenous biotin in samples can interfere with detection systems. Researchers should:

  • Implement avidin/streptavidin blocking steps before antibody application

  • Consider biotin-free detection alternatives when working with biotin-rich samples

  • Include biotin blocking controls in experimental design

• Cross-reactivity concerns: When studying SLAMF6 in multi-species contexts:

  • Verify species reactivity claims (e.g., human, mouse) experimentally

  • Test cross-reactivity with other SLAM family members, particularly those with high homology

  • Include genetic knockouts or knockdowns as definitive negative controls

• Clustering-dependent effects interpretation: Since SLAMF6 clustering is required for certain functions , researchers must distinguish between:

  • Effects of target engagement (epitope binding)

  • Effects of receptor clustering (signaling activation)

  • Effects of receptor blockade (preventing natural interactions)

Careful experimental design and appropriate controls help avoid misinterpretation of results when working with biotin-conjugated SLAMF6 antibodies.

How can researchers validate the specificity of biotin-conjugated SLAMF6 antibodies in their experimental systems?

Validating the specificity of biotin-conjugated SLAMF6 antibodies requires a multi-faceted approach:

• Genetic validation strategies:

  • Test antibody binding in SLAMF6 knockout or knockdown systems

  • Compare staining patterns in cell lines with confirmed high versus low SLAMF6 expression

  • Perform antibody testing in cells transfected with SLAMF6 versus empty vector controls

• Comprehensive antibody validation protocol:

  • Western blot: Confirm single band at expected molecular weight (111 kDa)

  • Immunoprecipitation: Verify pull-down of correctly sized protein

  • Flow cytometry: Compare staining pattern with alternative SLAMF6 antibody clones

  • Immunohistochemistry: Demonstrate expected tissue distribution pattern

• Competitive binding assays:

  • Pre-incubate with unconjugated antibody before adding biotin-conjugated version

  • Perform dose-dependent blocking with recombinant SLAMF6 protein

  • Compare binding profiles of Fab fragments versus whole antibodies

• Cross-reactivity assessment:

  • Test against related SLAM family members expressed in recombinant systems

  • Examine binding to species orthologs to confirm stated reactivity (human, mouse)

  • Evaluate performance in tissues with defined SLAMF6 expression patterns

• Technical validation approaches:

  • Verify biotin conjugation efficiency using streptavidin binding assays

  • Confirm antibody performance in multiple applications (ELISA, WB, flow cytometry)

  • Compare lot-to-lot consistency for reproducibility

Boster validates all antibodies on Western blot, immunohistochemistry, immunocytochemistry, immunofluorescence, and ELISA with known positive control and negative samples to ensure specificity and high affinity . Researchers should implement similar comprehensive validation in their specific experimental systems.

How are biotin-conjugated SLAMF6 antibodies being used to develop novel cancer immunotherapies?

Biotin-conjugated SLAMF6 antibodies are instrumental in developing next-generation cancer immunotherapies through several research avenues:

• Checkpoint blockade strategies: Research using SLAMF6-deficient T cells has demonstrated enhanced anti-tumor activity, suggesting SLAMF6 acts as an inhibitory checkpoint . Biotin-conjugated antibodies enable:

  • Screening of blocking antibody candidates through biotin-streptavidin-based binding inhibition assays

  • Evaluation of antibody-mediated SLAMF6 receptor clustering effects on T cell function

  • Development of biotin-conjugated anti-SLAMF6 antibodies as detection reagents in clinical trials

• Combinatorial immunotherapy approaches: SLAMF6 blockade effects can be further enhanced when combined with established immunotherapies . Researchers are using biotin-conjugated antibodies to:

  • Assess SLAMF6 expression before and after PD-1/CTLA-4 blockade

  • Develop multi-specific antibody constructs targeting SLAMF6 and other checkpoints

  • Study synergistic molecular mechanisms through multiplexed imaging and protein interaction studies

• CAR-T cell engineering: SLAMF6 knockout or blockade in adoptive T cell therapies shows promise. Biotin-conjugated antibodies facilitate:

  • Selection of SLAMF6-negative T cell populations for adoptive transfer

  • Monitoring SLAMF6 expression dynamics during CAR-T manufacturing

  • Studying the impact of SLAMF6 targeting on CAR-T persistence and function

• Biomarker development: SLAMF6 expression patterns and clustering dynamics may predict immunotherapy responses. Researchers utilize biotin-conjugated antibodies for:

  • Multiplex immunohistochemistry in tumor microenvironment studies

  • Flow cytometric analysis of circulating immune cells during immunotherapy

  • Development of companion diagnostics for SLAMF6-targeted therapies

These emerging approaches could lead to novel immunotherapeutic strategies targeting SLAMF6, potentially benefiting melanoma patients and those with other cancer types.

What technical advancements are improving the efficacy of biotin-conjugated SLAMF6 antibodies in research applications?

Recent technical advancements are significantly enhancing the utility of biotin-conjugated SLAMF6 antibodies:

• Site-specific biotinylation technologies:

  • Enzymatic biotinylation using BirA ligase for controlled biotin placement

  • Sortase-mediated conjugation enabling uniform antibody orientation

  • Click chemistry approaches allowing precise biotin-to-antibody ratios
    These advancements preserve epitope binding regions and reduce batch-to-batch variability compared to traditional random NHS-ester biotinylation.

• Enhanced detection systems:

  • Ultra-sensitive streptavidin polymers increasing signal amplification

  • Quantum dot-streptavidin conjugates providing photostable, narrow-emission signals

  • Tyramide signal amplification systems compatible with biotin-streptavidin interactions

• Multiplexing capabilities:

  • Conjugation to distinctly modified biotins (DSB-X biotin, photo-cleavable biotin)

  • Integration with cyclic immunofluorescence protocols for multi-parameter imaging

  • Compatibility with mass cytometry through metal-tagged streptavidin for high-dimensional analysis

• Structural optimization:

  • Development of recombinant antibody fragments with optimized biotin attachment sites

  • Strategic positioning of biotin molecules to maintain native protein interactions

  • Engineering of linker chemistry to reduce steric hindrance effects

• Quality control improvements:

  • Advanced purification techniques ensuring removal of unconjugated biotin

  • Quantitative assessment of biotin-to-antibody ratios for each production lot

  • Functional validation across multiple applications before commercial release

These technical advances are expanding the versatility and reliability of biotin-conjugated SLAMF6 antibodies across various research applications.

How can researchers utilize biotin-conjugated SLAMF6 antibodies in studying the cross-talk between SLAMF6 and other signaling pathways?

Investigating signaling cross-talk involving SLAMF6 requires sophisticated experimental approaches leveraging biotin-conjugated antibodies:

• Co-immunoprecipitation studies:

  • Use biotin-conjugated SLAMF6 antibodies with streptavidin beads to pull down intact signaling complexes

  • Analyze co-precipitated proteins by mass spectrometry to identify novel interaction partners

  • Compare complex formation under various stimulation conditions (TCR activation, cytokine treatment)
    This approach has revealed interactions between SLAMF6 and key signaling molecules like SAP and SHP-1 .

• Phosphoproteomics integration:

  • Stimulate cells with anti-CD3 and anti-SLAMF6 antibodies to activate specific pathways

  • Compare phosphorylation profiles of key signaling nodes (ERK, ZAP70, AKT, SRC)

  • Identify convergence points between SLAMF6 and TCR signaling cascades

• Molecular proximity analysis:

  • Employ biotin-conjugated antibodies in proximity ligation assays to visualize molecular interactions in situ

  • Use FRET-based approaches with biotin-conjugated primary and fluorophore-conjugated streptavidin

  • Apply BiFC (Bimolecular Fluorescence Complementation) to study direct protein interactions

• Functional cross-talk assessment:

  • Investigate how SLAMF6 clustering affects Rap1 activation using pull-down assays

  • Study the influence of SLAMF6 engagement on calcium flux and mitochondrial function

  • Examine transcriptional programs activated by SLAMF6 in presence/absence of TCR stimulation

• Temporal signaling dynamics:

  • Apply biotin-conjugated antibodies in time-course experiments to track signaling kinetics

  • Determine how SLAMF6 engagement modifies the duration of TCR-induced signaling

  • Assess feedback mechanisms regulating SLAMF6 expression and function after activation

These methodological approaches enable comprehensive investigation of how SLAMF6 integrates with broader immune signaling networks to regulate T cell function in health and disease.