borcs7 Antibody

What is BORCS7 and what cellular functions is it associated with?

BORCS7 (BLOC-1 Related Complex Subunit 7), previously known as C10orf32 or FLJ40752, is a component of the multisubunit BLOC-1-related complex (BORC) that regulates late endosomal/lysosomal positioning and function . Research has demonstrated that BORC plays crucial roles in coordinating the encounter and fusion of lysosomes with autophagosomes, suggesting BORCS7's involvement in cellular degradation pathways . Additionally, BORCS7 contributes to regulating late endosomal/lysosomal size through PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate mechanisms . Notably, BORCS7 has also been identified as a molecular risk factor in the 10q24.32 schizophrenia-associated locus, indicating potential roles in neuropsychiatric conditions .

What types of BORCS7 antibodies are available for research applications?

Based on current research resources, BORCS7 antibodies are predominantly available as rabbit polyclonal antibodies. These include affinity-purified antibodies suitable for multiple applications:

The antibodies are typically supplied in PBS (pH 7.2) with 40% glycerol and 0.02% sodium azide for stability .

What are the optimal experimental conditions for using BORCS7 antibodies in immunohistochemistry?

For optimal immunohistochemistry (IHC) applications with BORCS7 antibodies, researchers should follow these methodological guidelines:

• Dilution optimization: Use dilutions between 1:500-1:1000 for paraffin-embedded sections and 1:10-1:500 for frozen sections, with preliminary titration experiments recommended to determine optimal concentration for your specific tissue .

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective for BORCS7 detection, with 20 minutes at 95-98°C recommended.

• Detection systems: Both chromogenic (DAB) and fluorescent secondary detection systems are compatible with BORCS7 antibodies. For fluorescent detection, minimize exposure to light throughout the protocol.

• Controls: Always include positive controls (tissues known to express BORCS7) and negative controls (primary antibody omission and/or isotype controls) to validate staining specificity and troubleshoot potential background issues.

• Counterstaining: For brightfield microscopy, hematoxylin counterstaining provides good nuclear contrast against BORCS7 cytoplasmic staining patterns.

The validation data from antibody suppliers indicates successful IHC application across multiple normal and pathological human tissues, with predominantly cytoplasmic localization patterns consistent with BORCS7's role in endolysosomal pathways .

How should researchers optimize BORCS7 antibody protocols for immunocytochemistry/immunofluorescence (ICC-IF)?

For ICC-IF applications, consider these methodological approaches:

• Fixation methods: 4% paraformaldehyde (10-15 minutes at room temperature) typically preserves BORCS7 epitopes while maintaining cellular architecture. Methanol fixation (-20°C for 10 minutes) may provide alternative epitope accessibility.

• Antibody concentration: Recommended working concentration is 1-4 μg/ml, requiring dilution of stock solutions according to the specific antibody concentration . Optimization through serial dilutions is advised.

• Permeabilization: Use 0.1-0.3% Triton X-100 or 0.1% saponin in PBS for 5-10 minutes to facilitate antibody access to intracellular compartments while preserving antigenicity.

• Blocking conditions: 5-10% normal serum (from the same species as the secondary antibody) with 1% BSA in PBS for 30-60 minutes minimizes background staining.

• Co-localization studies: BORCS7 can be effectively co-stained with markers of late endosomes/lysosomes (LAMP1, LAMP2) or autophagy markers (LC3B) to investigate functional associations.

• Detection optimization: For weak signals, consider signal amplification systems like tyramide signal amplification, prolonged primary antibody incubation (overnight at 4°C), or higher antibody concentrations balanced against potential background increases.

Researchers should note that the cellular distribution pattern of BORCS7 typically presents as punctate cytoplasmic staining with enrichment in perinuclear regions, consistent with its endolysosomal localization .

How can researchers validate BORCS7 antibody specificity and resolve potential cross-reactivity issues?

Validating antibody specificity is critical for generating reliable research data. For BORCS7 antibodies, implement these validation approaches:

• Molecular weight verification: BORCS7 protein has a predicted molecular weight of approximately 12.7 kDa. In Western blot applications, confirm that the detected band corresponds to this expected size, accounting for potential post-translational modifications.

• Knockdown/knockout controls: Use siRNA/shRNA knockdown or CRISPR/Cas9 knockout models of BORCS7 to verify antibody specificity. Reduction or absence of signal in these models strongly supports antibody specificity.

• Peptide competition assays: Pre-incubate the BORCS7 antibody with excess immunizing peptide (when available from manufacturers) before application to samples. Specific signals should be significantly reduced or eliminated.

• Orthogonal detection methods: Compare results with alternative antibodies targeting different BORCS7 epitopes or with mRNA expression data from RT-PCR or RNA-seq studies.

• Recombinant protein controls: Use BORCS7 recombinant proteins as positive controls. Some manufacturers offer matching recombinant proteins or overexpression lysates for their antibodies .

If cross-reactivity is observed, consider these troubleshooting approaches:

• Increase antibody dilution to reduce non-specific binding

• Optimize blocking conditions with different blocking agents (BSA, normal serum, commercial blockers)

• Perform more stringent washing steps (increased duration, higher salt concentration)

• Use alternative antibody clones targeting different epitopes

Notably, some BORCS7 antibodies have been verified on protein arrays containing the target protein plus 383 other non-specific proteins to confirm specificity , providing an additional level of validation.

What methodological considerations should be implemented when studying BORCS7 in the context of lysosomal positioning and autophagy?

When investigating BORCS7's role in lysosomal positioning and autophagy, researchers should implement these specialized methodological approaches:

• Dynamic imaging protocols: Use live-cell imaging with fluorescently-tagged BORCS7 and/or lysosomal markers to track real-time changes in lysosomal positioning. Time-lapse microscopy with intervals of 15-30 seconds for 10-30 minutes can capture lysosomal movement dynamics.

• Colocalization analysis: Implement quantitative colocalization analysis between BORCS7 and:

  • Lysosomal markers (LAMP1, LAMP2)

  • Autophagosome markers (LC3, ATG proteins)

  • Other BORC components (BORCS5, etc.)

  Use Pearson's correlation coefficient, Manders' overlap coefficient, or object-based colocalization algorithms for quantitative assessment.

• Autophagy flux assessment: When studying BORCS7's role in autophagy:

  • Compare LC3-II levels with and without lysosomal inhibitors (bafilomycin A1, chloroquine)

  • Assess p62/SQSTM1 degradation as a measure of autophagic cargo processing

  • Use tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to discriminate between autophagosomes and autolysosomes

• Lysosomal size quantification: Since BORCS7 regulates late endosomal/lysosomal size through PIKfyve-dependent mechanisms , implement:

  • Automated morphometric analysis of lysosomal size using thresholding and particle analysis

  • Serial Z-stack imaging to capture the full three-dimensional nature of lysosomal structures

  • Time-course experiments following BORCS7 perturbation to track progressive changes in lysosomal morphology

• PIKfyve pathway manipulation: To study the relationship between BORCS7 and phosphatidylinositol-3,5-bisphosphate:

  • Use PIKfyve inhibitors (e.g., apilimod, YM201636) alongside BORCS7 manipulation

  • Implement phosphoinositide probes to monitor PI(3,5)P2 levels in response to BORCS7 alterations

  • Consider epistasis experiments with PIKfyve and BORCS7 manipulations to establish pathway relationships

When publishing results, quantitative analysis of multiple cells (n>30) across at least three independent experiments is recommended for statistical robustness.

How should researchers design experiments to investigate BORCS7's role in schizophrenia and neuropsychiatric disorders?

Research has identified BORCS7 as a molecular risk factor in the 10q24.32 schizophrenia-associated locus . When designing experiments to investigate this connection, consider these specialized methodological approaches:

• Genotype-expression correlation studies:

  • Analyze BORCS7 expression levels in post-mortem brain samples stratified by 10q24.32 risk allele status

  • Implement allele-specific expression analysis to detect cis-regulatory effects

  • Consider brain region-specific analysis, with particular focus on regions implicated in schizophrenia pathophysiology

• Cellular model systems:

  • Generate isogenic neural cell lines (using CRISPR/Cas9) with and without schizophrenia-associated variants

  • Develop patient-derived iPSC models differentiated into relevant neural cell types

  • Compare BORCS7 expression, subcellular localization, and functional effects between risk and non-risk genetic backgrounds

• Transcriptional regulation analysis:

  • Investigate potential linkage between BORCS7 and the BORCS7-ASMT readthrough transcript, which has been identified as a non-coding RNA gene

  • Implement chromosome conformation capture techniques (3C, 4C, Hi-C) to assess potential long-range interactions affecting BORCS7 regulation

  • Conduct chromatin immunoprecipitation (ChIP) to identify transcription factors mediating risk variant effects

• Functional pathway analysis:

  • Investigate if schizophrenia-associated BORCS7 variants alter endolysosomal or autophagic pathways in neuronal models

  • Assess potential impacts on neurodevelopmental processes, synaptic function, or neuronal connectivity

  • Consider interactions with other schizophrenia risk genes operating in related cellular pathways

• Translational approaches:

  • Develop high-throughput screening methods to identify compounds that normalize aberrant BORCS7 expression or function

  • Investigate potential biomarker applications in peripheral samples (blood, induced neurons) from individuals with schizophrenia

When designing these experiments, researchers should account for the complex genetic architecture of schizophrenia, potentially implementing polygenic risk score analyses alongside specific BORCS7 investigations.

What methodological approaches are recommended for investigating protein-protein interactions between BORCS7 and other BORC complex components?

Investigating BORCS7's interactions within the BORC complex requires specialized methodological approaches:

• Co-immunoprecipitation (Co-IP) optimization:

  • Use BORCS7 antibodies for immunoprecipitation followed by Western blotting for other BORC components

  • Consider reversing the procedure (IP other BORC components, blot for BORCS7)

  • Optimize lysis conditions to preserve protein-protein interactions (mild non-ionic detergents like 0.5-1% NP-40 or 0.5% Triton X-100)

  • Include appropriate controls (IgG control, input samples, and when possible, BORCS7 knockout/knockdown controls)

• Proximity labeling approaches:

  • Implement BioID or APEX2 proximity labeling by fusing these enzymes to BORCS7

  • Express the fusion protein in relevant cell types to biotin-label proximal proteins

  • Identify interacting partners through streptavidin pulldown followed by mass spectrometry

  • Compare results with other BORC components' interactomes to identify shared and unique interactors

• Fluorescence-based interaction assays:

  • Förster resonance energy transfer (FRET) between fluorescently-tagged BORCS7 and other BORC components

  • Bimolecular fluorescence complementation (BiFC) to visualize interaction sites within cells

  • Fluorescence correlation spectroscopy (FCS) to analyze binding dynamics and affinities

• Structural biology approaches:

  • Recombinant expression and purification of BORCS7 for structural studies

  • X-ray crystallography or cryo-electron microscopy of BORCS7 alone or in complex with binding partners

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

• Functional interaction studies:

  • Generate domain deletion or point mutation constructs to map specific interaction domains

  • Assess the impact of these mutations on complex formation and function

  • Implement rescue experiments following BORCS7 depletion with wild-type versus mutant constructs

When publishing interaction data, it's recommended to validate key findings using at least two independent methods (e.g., Co-IP plus proximity labeling) to increase confidence in the results.

How can researchers effectively use BORCS7 antibodies in tissue microarrays and high-throughput screening applications?

For tissue microarray (TMA) and high-throughput applications with BORCS7 antibodies, implement these specialized methodological considerations:

• TMA protocol optimization:

  • Standardize fixation and processing conditions across all TMA samples

  • Include positive and negative control cores on each TMA block

  • Optimize antigen retrieval methods specifically for the TMA format

  • Consider multiple cores per case to account for tumor heterogeneity

  • Implement automated staining platforms for consistency across large sample sets

• Signal detection and quantification:

  • Use digital pathology systems for standardized image acquisition

  • Implement computational image analysis for objective quantification:

    • Measure both staining intensity and percentage of positive cells

    • Develop algorithms to distinguish cellular compartment-specific staining

    • Consider machine learning approaches for pattern recognition

• Multiplexed detection strategies:

  • Sequential immunofluorescence with antibody stripping between rounds

  • Implement spectral unmixing for simultaneous detection of multiple markers

  • Consider mass cytometry (CyTOF) or imaging mass cytometry approaches for highly multiplexed analyses

• Quality control measures:

  • Include replicate samples across multiple TMAs to assess reproducibility

  • Implement positive signal normalization across batches

  • Consider automated systems to minimize human variability

• Data integration approaches:

  • Correlate BORCS7 expression patterns with:

    • Clinical parameters and outcomes

    • Expression of other BORC complex components

    • Lysosomal and autophagy pathway markers

Based on the validated applications of available BORCS7 antibodies , these approaches are technically feasible for most research-grade antibodies, though each application should be independently validated.

What are the critical considerations when analyzing contradictory BORCS7 expression data across different experimental platforms?

When faced with contradictory BORCS7 expression data across experimental platforms (e.g., immunohistochemistry vs. Western blot vs. RNA-seq), implement these analytical approaches:

• Technical validation and troubleshooting:

  • Verify antibody specificity using methods described in question 3.1

  • Assess potential isoform-specific detection differences between antibodies targeting different epitopes

  • Consider differences in detection sensitivity between methods

  • Evaluate post-translational modifications that might affect antibody binding

• Biological variability analysis:

  • Determine if inconsistencies correlate with specific sample characteristics (tissue type, disease state, genetic background)

  • Analyze potential biological mechanisms that could explain differential expression:

    • Cell type-specific expression patterns

    • Subcellular localization differences

    • Condition-dependent expression regulation

• Data integration strategies:

  • Implement meta-analysis techniques to identify robust signals across platforms

  • Consider Bayesian integration approaches to weigh evidence from multiple sources

  • Use orthogonal validation with independent methods and reagents

• Specific contradictions to consider:

  • mRNA vs. protein discrepancies: Analyze potential post-transcriptional regulation

  • Antibody vs. antibody differences: Consider epitope accessibility in different experimental contexts

  • In vitro vs. in vivo differences: Evaluate microenvironmental factors affecting expression

• Standardization approaches:

  • Implement absolute quantification methods where possible

  • Use identical reference standards across platforms

  • Consider spike-in controls for cross-platform normalization

When reporting contradictory findings, researchers should present all data transparently with detailed methodological descriptions to allow readers to evaluate potential sources of discrepancy.

How might advanced genome editing approaches be used to investigate BORCS7 function in cellular models?

Emerging CRISPR-based technologies offer sophisticated approaches for studying BORCS7 function:

• Precision genome editing strategies:

  • CRISPR base editors for introducing precise single nucleotide variants associated with schizophrenia risk

  • Prime editing for "scarless" introduction of specific mutations

  • Homology-directed repair for knock-in of reporter tags or functional domains

• Regulatory element manipulation:

  • CRISPRi (dCas9-KRAB) for targeted repression of BORCS7 expression

  • CRISPRa (dCas9-VP64/p65/HSF1) for upregulation studies

  • CRISPR engineering of enhancers or promoters to study cis-regulatory mechanisms

• Spatiotemporal control systems:

  • Optogenetic or chemically-inducible Cas9 systems for temporal control of BORCS7 disruption

  • Cell type-specific Cas9 expression for tissue-selective editing

  • Inducible degradation systems (e.g., AID, dTAG) for acute protein depletion studies

• High-throughput functional genomics:

  • CRISPR screens targeting potential BORCS7 interactors or regulatory factors

  • Perturb-seq combining CRISPR perturbations with single-cell RNA-seq

  • CRISPR tiling screens across BORCS7 locus to identify functional regulatory elements

• Disease modeling applications:

  • Introduction of patient-specific variants into isogenic backgrounds

  • Correction of variants in patient-derived cells

  • Combinatorial editing of multiple schizophrenia risk loci to study epistatic interactions

These approaches go beyond conventional knockout strategies to provide nuanced insights into BORCS7 function and regulation, particularly in the context of its roles in lysosomal positioning, autophagy, and potential contributions to neuropsychiatric disorders.

What methodological considerations should researchers address when developing BORCS7-targeted therapeutic approaches?

For researchers exploring BORCS7 as a potential therapeutic target, especially in contexts like schizophrenia where it has been identified as a risk factor , these methodological considerations are critical:

• Target validation approaches:

  • Establish clear disease-associated phenotypes linked to BORCS7 dysfunction

  • Validate these phenotypes across multiple model systems (cell lines, primary cells, animal models)

  • Determine if normalization of BORCS7 expression/function rescues disease phenotypes

  • Identify specific protein domains or interactions most suitable for therapeutic targeting

• Therapeutic modality selection:

  • Small molecule approaches targeting BORCS7 protein-protein interactions

  • Antisense oligonucleotides or siRNA for expression modulation

  • Protein replacement strategies for loss-of-function scenarios

  • Gene therapy approaches for long-term correction

• Screening methodology design:

  • Develop robust high-throughput assays with disease-relevant readouts

  • Implement counter-screens to assess specificity versus other BORC components

  • Consider phenotypic screening approaches focused on normalizing cellular dysfunctions

  • Develop appropriate in vitro to in vivo translation strategies

• Safety and off-target assessment:

  • Comprehensive profiling of BORCS7 expression across tissues

  • Toxicity assessment in systems with high BORCS7 expression

  • Analysis of effects on other BORC complex members and lysosomal pathways

  • Long-term studies to evaluate compensatory mechanisms

• Biomarker development:

  • Identify measurable indicators of BORCS7 pathway engagement

  • Develop assays to quantify target engagement in accessible tissues

  • Establish correlation between biomarker changes and functional outcomes