SLITRK5 Antibody, Biotin conjugated

What is SLITRK5 and why is it significant in neuroscience research?

SLITRK5 is a membrane-localized protein belonging to the SLITRK family with a reported length of 958 amino acid residues and a molecular mass of 107.5 kDa in humans . It contains leucine-rich repeat (LRR) domains that mediate protein-protein interactions and is highly expressed in the cerebral cortex, with lower expression levels in the spinal cord and medulla . Its significance stems from its role in neuronal development, chemical synaptic transmission, and its emergence as a candidate gene for neuropsychiatric disorders . Recent studies have revealed that SLITRK5 functions as a critical mediator of BDNF-dependent TrkB receptor trafficking and signaling, suggesting its importance in neurotrophin signaling pathways that regulate neuronal survival, differentiation, and plasticity .

What are the structural characteristics of SLITRK5 protein?

SLITRK5 protein contains two leucine-rich repeat (LRR) domains in its extracellular portion, with the first LRR domain (LRR1) mediating interactions with TrkB receptors . The protein undergoes post-translational modifications, notably glycosylation, which may affect its function and interactions . SLITRK5 is localized to the cell membrane and can exist in up to two different isoforms in humans . The protein's structure enables it to engage in both trans-synaptic interactions with proteins like PTPδ and cis-interactions with receptors like TrkB, with these interaction preferences being modulated by factors such as BDNF stimulation .

How specific are commercially available SLITRK5 antibodies?

Commercial SLITRK5 antibodies demonstrate varying degrees of specificity. Research has shown that certain SLITRK5 antibodies can distinguish SLITRK5 from other SLITRK family members (SLITRK1-3), indicating good isoform specificity . The specificity is particularly important given SLITRK5's LRR domain similarity to other family members. When selecting an antibody, researchers should consider the recognized epitope location, as antibodies targeting the first LRR domain may detect interactions with binding partners like TrkB and PTPδ . For optimal specificity validation, co-immunoprecipitation experiments comparing SLITRK5 with other family members are recommended, as demonstrated in published research where SLITRK5-TrkB interactions were shown to be specific and not observed with SLITRK1-3 .

What are the primary applications for SLITRK5 antibodies in research?

SLITRK5 antibodies are utilized across multiple research applications with Western blot being the most common . Other significant applications include:

• ELISA - Particularly suitable for biotin-conjugated antibodies for quantitative detection

• Immunocytochemistry - For cellular localization studies

• Immunofluorescence - For co-localization studies with interacting partners

• Immunohistochemistry - For tissue expression pattern analysis

These applications have been instrumental in characterizing SLITRK5's expression patterns, protein-protein interactions, and its role in neuronal signaling pathways . In particular, immunofluorescence techniques have revealed the BDNF-dependent shift in Slitrk5 localization from trans-synaptic interactions with PTPδ to cis-interactions with TrkB receptors in endosomal structures .

How does biotin conjugation affect SLITRK5 antibody performance in different assays?

Biotin conjugation of SLITRK5 antibodies offers several methodological advantages while potentially introducing specific considerations for experimental design. The biotin-streptavidin system provides signal amplification that can enhance detection sensitivity in ELISA and other assays . In co-localization studies, biotin-conjugated antibodies allow for flexible secondary detection strategies and multi-color imaging protocols when examining SLITRK5 interactions with partners like TrkB and PTPδ .

• Conjugation ratio optimization - Excessive biotinylation may interfere with epitope recognition

• Blocking protocols - Endogenous biotin must be blocked, especially in brain tissue with high biotin content

• Buffer compatibility - Some detergents may affect biotin-streptavidin interactions

For quantitative ELISA applications, biotin-conjugated SLITRK5 antibodies show particular utility, with detection limits in the nanogram range for recombinant SLITRK5 protein .

What are the optimal protocols for visualizing SLITRK5-TrkB interactions using immunofluorescence?

Visualizing SLITRK5-TrkB interactions requires specialized protocols that account for the dynamic, BDNF-dependent nature of these interactions. Based on published research methodologies, the following approach has proven effective:

• Sample preparation:

  • For primary neurons: Culture on poly-L-lysine coated coverslips for 14-21 DIV

  • For cell lines: Co-transfect with tagged SLITRK5 (FLAG/GFP) and TrkB constructs

• Stimulation conditions:

  • Serum starvation (0% FBS) for 4-6 hours to reduce basal interactions

  • BDNF treatment (25-100 ng/ml) for 5-30 minutes to induce interactions

• Fixation and immunostaining:

  • Brief (10 min) 4% paraformaldehyde fixation to preserve membrane interactions

  • Permeabilization with 0.1% Triton X-100

  • Primary antibodies: anti-SLITRK5 (biotin-conjugated) and anti-TrkB

  • Secondary detection: streptavidin-conjugated fluorophore and species-specific antibody

• Advanced imaging:

  • Structured illumination microscopy for sub-diffraction resolution

  • 3D reconstruction using software like IMARIS

  • Co-localization analysis with ImageJ co-localization highlighter

This approach has successfully demonstrated that BDNF stimulation shifts SLITRK5 from trans-interactions with PTPδ to cis-interactions with TrkB in punctate endosomal structures .

How can researchers control for specificity when using SLITRK5 antibodies in protein interaction studies?

When investigating SLITRK5 protein interactions, controlling for specificity is crucial due to the structural similarities with other SLITRK family members. Recommended controls include:

• Family member controls:

  • Include other SLITRK family members (SLITRK1-3) in parallel experiments

  • Compare interaction patterns between family members and potential binding partners

• Domain-specific controls:

  • Utilize chimeric constructs swapping LRR domains between SLITRK family members

  • LRR1 domain from SLITRK5 is specific for TrkB interactions and can be used as a positive control

• Biochemical validation:

  • Competitive binding assays with purified protein domains

  • Dissociation studies using increasing concentrations of potential competitors

• Negative controls:

  • Test interactions with unrelated receptors (e.g., TrkC showed no interaction with SLITRK5)

  • Use kinase inhibitors (e.g., K252a for TrkB) to confirm interaction dependencies

Published research has demonstrated that co-immunoprecipitation experiments with these controls can effectively distinguish specific SLITRK5-TrkB interactions from non-specific binding .

What experimental approaches best demonstrate SLITRK5's role in TrkB receptor trafficking?

Investigating SLITRK5's role in TrkB receptor trafficking requires specialized techniques that capture dynamic membrane protein movements. Based on successful published approaches, researchers should consider:

• Receptor recycling assays:

  • Surface biotinylation followed by cleavage and re-biotinylation to track recycling rates

  • Comparing wild-type and SLITRK5-knockout/knockdown models for quantitative differences

• Endosomal co-localization studies:

  • Structured illumination microscopy to visualize SLITRK5, TrkB, and Rab11-positive recycling endosomes

  • Triple co-localization analysis with Rab11-FIP3 (a Rab11 effector protein)

• Live-cell imaging:

  • Pulse-chase experiments with pH-sensitive fluorophore-tagged TrkB receptors

  • TIRF microscopy to visualize membrane-proximal trafficking events

• Trafficking complex isolation:

  • Immunoprecipitation of SLITRK5 followed by identification of associated trafficking proteins

  • Analysis of protein complexes before and after BDNF stimulation

These approaches have demonstrated that SLITRK5 mediates optimal targeting of TrkB receptors to Rab11-positive recycling endosomes through recruitment of Rab11-FIP3, revealing a mechanism by which SLITRK5 regulates BDNF-dependent TrkB trafficking and signaling .

How can SLITRK5 antibodies be used to investigate the relationship between SLITRK5 dysfunction and neuropsychiatric disorders?

SLITRK5 antibodies provide valuable tools for investigating the link between SLITRK5 dysfunction and neuropsychiatric disorders through several methodological approaches:

• Expression analysis in patient-derived samples:

  • Quantitative immunohistochemistry in post-mortem brain tissues

  • Western blot analysis of SLITRK5 expression levels in patient-derived neuronal cultures

• Functional interaction studies:

  • Co-immunoprecipitation experiments to assess altered binding to TrkB or PTPδ in disease models

  • Analysis of BDNF-dependent trafficking in neurons derived from patients or animal models

• Genetic variant characterization:

  • Generation of disease-associated SLITRK5 mutations

  • Antibody-based assessment of mutant protein localization and interaction profiles

• Pathway analysis:

  • Phospho-specific antibodies to track downstream signaling abnormalities

  • Biotin-conjugated SLITRK5 antibodies for pull-down of novel interaction partners in disease states

These approaches leverage the specificity of SLITRK5 antibodies to interrogate the molecular mechanisms by which SLITRK5 dysfunction may contribute to neuropsychiatric disorders, building on studies that have identified SLITRK5 as a candidate gene for these conditions .

What are the optimal sample preparation methods for detecting SLITRK5 in different tissue types?

Sample preparation for SLITRK5 detection varies by tissue type and intended application. Based on research practices, the following protocols are recommended:

For brain tissue:

• Fresh-frozen sections (10-20 μm) yield better epitope preservation than formalin-fixed tissues

• Antigen retrieval using citrate buffer (pH 6.0) at 95°C for 15-20 minutes improves detection

• Permeabilization with 0.2-0.3% Triton X-100 enables detection of intracellular epitopes

• Pre-blocking with 5-10% normal serum from the species of secondary antibody production

For cultured neurons:

• Fixation with 4% paraformaldehyde for 10-15 minutes at room temperature

• Mild permeabilization (0.1% Triton X-100 for 5-10 minutes) to preserve membrane structure

• BSA (3-5%) with 0.1% Tween-20 as blocking and antibody dilution buffer

• For biotin-conjugated antibodies, additional blocking of endogenous biotin is essential

The high expression of SLITRK5 in cerebral cortex requires careful titration of antibody concentrations, with 1:200-1:500 dilutions typically providing optimal signal-to-noise ratios for immunohistochemical applications .

What are the key considerations for quantitative analysis of SLITRK5 using biotin-conjugated antibodies in ELISA?

Quantitative analysis of SLITRK5 using biotin-conjugated antibodies in ELISA requires attention to several methodological details:

• Assay design considerations:

  • Sandwich ELISA using a capture antibody against a different epitope than the biotin-conjugated detection antibody

  • Recombinant SLITRK5 protein standards covering 0.1-100 ng/ml range

  • Streptavidin-HRP dilution optimization (typically 1:5000-1:20000)

• Sample preparation:

  • Tissue lysates require detergent optimization (typically 0.5-1% NP-40 or Triton X-100)

  • Cerebrospinal fluid should be diluted 1:2-1:5 in blocking buffer

  • Cell culture supernatants may require concentration for detection

• Data analysis:

  • Four-parameter logistic regression for standard curve fitting

  • Normalization to total protein concentration for tissue samples

  • Technical triplicates with CV < 15% for reliable quantification

• Validation controls:

  • Spike-and-recovery experiments to assess matrix effects

  • Parallelism testing between recombinant and endogenous SLITRK5

  • Comparison with SLITRK5 detection by Western blot for cross-validation

These methodological considerations support accurate and reproducible quantification of SLITRK5 in experimental samples when using biotin-conjugated antibodies in ELISA applications .

How can researchers troubleshoot non-specific binding issues with SLITRK5 antibodies?

Non-specific binding is a common challenge when working with SLITRK5 antibodies. The following troubleshooting strategies address specific issues:

• High background in immunohistochemistry/immunofluorescence:

  • Increase blocking duration (2-4 hours) with 5% normal serum and 1-2% BSA

  • Include 0.1-0.3% Triton X-100 in blocking solution to reduce hydrophobic interactions

  • For biotin-conjugated antibodies, use avidin/biotin blocking kits to eliminate endogenous biotin signals

  • Titrate primary antibody concentration (start with 1:500 and adjust as needed)

• Multiple bands in Western blot:

  • Increase stringency of wash buffers (0.1-0.3% Tween-20 in TBS)

  • Use gradient gels to better separate SLITRK5 from similar molecular weight proteins

  • Validate specificity with SLITRK5 knockout/knockdown controls

  • Consider native vs. reducing conditions to account for glycosylation effects

• False positive co-immunoprecipitation:

  • Include isotype control antibodies in parallel experiments

  • Perform reverse co-IP (immunoprecipitate with anti-TrkB and detect SLITRK5)

  • Use crosslinking agents of varying length to distinguish direct vs. indirect interactions

  • Compare results across multiple detergent conditions (NP-40, CHAPS, Triton X-100)

These troubleshooting approaches have been successfully applied in SLITRK5 research to distinguish specific signals from background and ensure reliable results .