OSBPL7 Antibody, Biotin conjugated

What is OSBPL7 and what cellular functions does it participate in?

OSBPL7 (Oxysterol Binding Protein-Like 7) is a 95.4 kDa protein (842 amino acids) belonging to the OSBP protein family with subcellular localization in the cell membrane, endoplasmic reticulum, and cytoplasm . It functions primarily in cholesterol metabolism and homeostasis . Recent research has demonstrated OSBPL7's critical involvement in ABCA1-dependent cholesterol efflux, particularly in kidney cells, where it has protective effects against renal disease progression . Additionally, OSBPL7 participates in macroautophagy pathways, specifically in the selective autophagy of protein aggregates (aggrephagy) and xenophagy (clearance of intracellular pathogens) . The protein has emerged as a novel lipid transfer protein that interacts with the autophagy machinery, complementing other lipid transfer mechanisms in autophagosome formation .

How do biotin-conjugated antibodies differ from unconjugated antibodies in OSBPL7 detection?

Biotin-conjugated anti-OSBPL7 antibodies provide significant advantages over unconjugated antibodies for specific detection methodologies. The biotin-streptavidin system exhibits exceptional binding affinity (Kd ≈ 10^-15 M), providing robust signal amplification without introducing steric hindrance that might affect epitope recognition. For OSBPL7 detection, this conjugation enables:

• Enhanced sensitivity in detecting low expression levels of OSBPL7 in tissues like intestinal epithelium

• Compatibility with multiple detection systems (colorimetric, fluorescent, chemiluminescent) without changing the primary antibody

• Reduced background when examining OSBPL7's distribution across subcellular compartments

• Ability to perform multiplex staining to simultaneously visualize OSBPL7 and its interaction partners

What is the specificity profile of commercially available biotin-conjugated anti-OSBPL7 antibodies?

Available biotin-conjugated anti-OSBPL7 antibodies, such as ABIN7162408, specifically target the N-terminal region (amino acids 1-249) of human OSBPL7 . This region is distinct from the sterol-binding pocket (which includes residues like Lys636, Ile641, Val616, and Val618) . The specificity profile indicates:

When selecting these antibodies, researchers should verify the antibody's validation data against positive and negative controls to ensure specificity for their particular experimental system .

What are the optimal protocols for using biotin-conjugated anti-OSBPL7 antibodies in ELISA assays?

For optimal ELISA performance with biotin-conjugated anti-OSBPL7 antibodies, the following methodological approach is recommended:

• Plate Preparation:

  • Coat high-binding 96-well plates with capture antibody against OSBPL7 (1-5 μg/mL) in carbonate buffer (pH 9.6)

  • Incubate overnight at 4°C, then wash 3× with PBS-T (0.05% Tween-20)

  • Block with 2-5% BSA in PBS for 1-2 hours at room temperature

• Sample Preparation:

  • For cell lysates: Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Dilute samples in blocking buffer to fall within the linear range of detection

  • Include known concentration standards for quantification

• Detection Procedure:

  • Add samples and incubate for 2 hours at room temperature

  • Wash 5× with PBS-T

  • Apply biotin-conjugated anti-OSBPL7 antibody (ABIN7162408) at 1:1000-1:5000 dilution and incubate for 1-2 hours

  • Wash 5× with PBS-T

  • Add streptavidin-HRP (1:5000-1:10000) and incubate for 30-60 minutes

  • Wash 5× with PBS-T

  • Develop with TMB substrate and stop with 2N H₂SO₄

  • Read absorbance at 450nm with 570nm reference

This protocol has demonstrated sensitivity for detecting OSBPL7 concentrations as low as 0.1 ng/mL in research settings . For quantitative analysis, it's essential to include a standard curve using recombinant OSBPL7 protein (such as those produced in HEK-293 cells) .

How can biotin-conjugated anti-OSBPL7 antibodies be employed to study OSBPL7's role in autophagy pathways?

Biotin-conjugated anti-OSBPL7 antibodies offer versatile approaches to investigate OSBPL7's emerging role in autophagy:

• Co-localization Studies:

  • Use biotin-conjugated anti-OSBPL7 antibodies followed by streptavidin-fluorophore conjugates

  • Counter-stain with markers for autophagosome formation (LC3B), lysosomal compartments (LAMP1/2), or lipid droplets

  • Analyze using confocal microscopy to determine spatial relationships during autophagy progression

• Pull-down Assays:

  • Employ the biotin-conjugated antibody to immunoprecipitate OSBPL7 and its associated proteins

  • Analyze interaction with LC3B-I specifically (as OSBPL7 has been shown to interact with the unlipidated form)

  • Examine changes in interaction profiles under autophagy-inducing conditions (starvation, rapamycin treatment)

• Proximity Ligation Assay (PLA):

  • Combine biotin-conjugated anti-OSBPL7 with antibodies against putative interaction partners

  • Visualize direct protein-protein interactions within 40nm distance using streptavidin-oligonucleotide and rolling circle amplification

  • Quantify interaction events during different stages of autophagy

Recent research has revealed that OSBPL7 knockdown impairs LC3B recruitment to Salmonella and disrupts aggrephagy (clearance of protein aggregates), indicating its essential role in selective autophagy pathways . Researchers should design experiments to distinguish between OSBPL7's role in different forms of selective autophagy versus its functions in bulk autophagy.

What controls should be included when validating results from experiments using biotin-conjugated anti-OSBPL7 antibodies?

Rigorous validation requires multiple controls to ensure reliable interpretation of results:

When studying OSBPL7's interaction with autophagy proteins like LC3B, researchers should include controls treating cells with autophagy modulators (e.g., rapamycin, bafilomycin A1) to confirm that observed interactions respond appropriately to autophagy induction or inhibition .

How can biotin-conjugated anti-OSBPL7 antibodies be utilized to investigate the OSBPL7-ABCA1 regulatory axis in kidney disease models?

Research has identified OSBPL7 as a therapeutic target for kidney disease through its role in ABCA1-dependent cholesterol efflux . Biotin-conjugated anti-OSBPL7 antibodies can be strategically employed to investigate this pathway:

• Mechanistic Studies:

  • Use proximity ligation assays (PLA) with biotin-conjugated anti-OSBPL7 and anti-ABCA1 antibodies to quantify their spatial relationship in kidney cells under normal and disease conditions

  • Perform co-immunoprecipitation experiments to isolate OSBPL7-associated protein complexes, followed by western blotting for ABCA1 and other cholesterol regulatory proteins

  • Immunohistochemistry on kidney sections from disease models (Adriamycin-induced nephropathy, Alport Syndrome) to track changes in OSBPL7 expression and localization

• Therapeutic Response Monitoring:

  • Track changes in OSBPL7-ABCA1 association in response to 5-arylnicotinamide compounds that target OSBPL7's sterol binding pocket

  • Develop ELISA-based screening assays to identify compounds that modulate OSBPL7-ABCA1 interaction

  • Quantify changes in OSBPL7 membrane localization following treatment with compounds that normalize proteinuria

• Structural Insights:

  • Use biotin-conjugated antibodies in conjunction with proximity labeling techniques to map conformational changes in OSBPL7 upon ligand binding

  • Investigate whether therapeutic compounds induce changes in OSBPL7's interaction landscape

Research indicates that while OSBPL7 and ABCA1 functionally interact in the cholesterol efflux pathway, direct physical interaction between these proteins has not been consistently detected in co-immunoprecipitation experiments . This suggests the relationship may involve intermediate proteins or transient interactions that require specialized detection methods.

What methodological approaches can resolve contradictory findings regarding OSBPL7's protein-protein interactions?

Published literature contains some contradictions regarding OSBPL7's protein interaction partners. These can be resolved through careful methodological approaches:

• Interaction Context Dependency:

  • Some studies show OSBPL7 interacts with LC3B-I (unlipidated form) , while others found no direct interaction with certain partners like ABCA1

  • Resolution approach: Conduct interaction studies under multiple cellular conditions (nutrient starvation, cholesterol loading/depletion, autophagy induction) to identify context-dependent interactions

• Interaction Kinetics:

  • Many interactions may be transient or of low affinity

  • Resolution approach: Employ crosslinking strategies before immunoprecipitation with biotin-conjugated anti-OSBPL7 antibodies to capture fleeting interactions

• Spatial Resolution:

  • Immunofluorescence studies sometimes yield different results than biochemical approaches

  • Resolution approach: Combine super-resolution microscopy (STORM/PALM) using biotin-streptavidin detection systems with proximity labeling techniques (BioID, APEX) to map spatial interactions with nanometer precision

• Isoform-Specific Interactions:

  • OSBPL7 has two reported isoforms that may have different interaction profiles

  • Resolution approach: Design isoform-specific detection strategies and validate antibody recognition of different isoforms

A comprehensive approach combining multiple methodologies can help reconcile contradictory findings. For example, while conventional co-immunoprecipitation failed to detect OSBPL7-ABCA1 interaction , the functional relationship between these proteins suggests alternative techniques like proximity labeling or FRET-based approaches may reveal transient or proximity-based associations.

How can researchers optimize detection of OSBPL7 in subcellular fractions during autophagy studies?

OSBPL7 exhibits dynamic subcellular localization during autophagy processes, requiring optimized detection protocols:

• Subcellular Fractionation Protocol:

  • Begin with differential centrifugation to separate major compartments (nuclear, cytosolic, membrane, organelle)

  • Further purify autophagosomal fractions using density gradient centrifugation

  • For each fraction, normalize loading by fraction-specific markers:

    • Cytosol: GAPDH or tubulin

    • Membranes: Na⁺/K⁺-ATPase

    • ER: Calnexin or PDI

    • Autophagosomes: LC3-II

    • Lysosomes: LAMP1/2

• Detection Optimization:

  • For Western blotting: Use biotin-conjugated anti-OSBPL7 followed by streptavidin-HRP at 1:10,000 dilution

  • For immunofluorescence microscopy: Apply biotin-conjugated anti-OSBPL7 (1:200-1:500) followed by streptavidin-fluorophore conjugates

  • For electron microscopy: Employ gold-conjugated streptavidin following biotin-antibody labeling

• Dynamic Tracking:

  • Monitor OSBPL7 redistribution during autophagy progression using time-course experiments

  • Quantify relative enrichment in different compartments using densitometry (western blots) or fluorescence intensity measurements (microscopy)

Research has shown that OSBPL7 is required for proper LC3B recruitment to Salmonella and for aggregate clearance in aggrephagy , suggesting its dynamic movement between cellular compartments during these processes. When designing fractionation experiments, special attention should be paid to membrane contact sites between the ER and forming autophagosomes, as these may be sites of OSBPL7 action in its role as a lipid transfer protein.

What are common pitfalls when using biotin-conjugated anti-OSBPL7 antibodies and how can they be addressed?

Several technical challenges can arise when working with biotin-conjugated anti-OSBPL7 antibodies:

When investigating OSBPL7's role in lipid transfer during autophagy, researchers should be particularly attentive to fixation methods, as these can disrupt membrane structures and lipid distributions, potentially affecting observed localization patterns .

How should researchers interpret changes in OSBPL7 localization patterns in relation to autophagy dynamics?

Interpretation of OSBPL7 localization requires careful consideration of autophagy dynamics:

• Baseline Distribution:

  • Under normal conditions, OSBPL7 shows distribution between cytosol, plasma membrane, and perinuclear ER

  • Punctate structures may represent association with lipid droplets or vesicular compartments

• Early Autophagy Induction Changes:

  • Significant redistribution to forming isolation membranes may indicate involvement in early autophagosome formation

  • Co-localization with early autophagy markers (ULK1, WIPI2) suggests role in initiation

  • OSBPL7 may facilitate lipid transfer at this stage to support membrane expansion

• Late Autophagy Association:

  • Association with LC3B-positive structures indicates incorporation into mature autophagosomes

  • Persistence on autophagosomes during fusion with lysosomes suggests roles beyond formation

• Quantitative Assessment:

  • Measure Pearson's correlation coefficient between OSBPL7 and various autophagy markers

  • Track temporal changes in colocalization during autophagy progression

  • Compare wild-type patterns with OSBPL7 mutants affecting sterol binding capacity

Research has demonstrated that OSBPL7 knockdown impairs both xenophagy (LC3B recruitment to Salmonella) and aggrephagy (clearance of protein aggregates) , suggesting it functions at critical stages of selective autophagy. When interpreting localization data, researchers should distinguish between OSBPL7's role in bulk autophagy versus selective autophagy, as these pathways may involve different recruitment mechanisms and timing.

How can researchers distinguish between direct and indirect effects of OSBPL7 manipulation on autophagy pathways?

Differentiating direct from indirect effects requires multi-faceted experimental approaches:

• Temporal Analysis:

  • Perform time-course experiments after OSBPL7 manipulation (knockdown/overexpression)

  • Immediate effects (0-4 hours) more likely represent direct mechanisms

  • Delayed effects (>12 hours) may indicate secondary consequences

• Domain Mutation Studies:

  • Generate OSBPL7 constructs with mutations in:

    • Lipid-binding domain (e.g., mutations at Lys636, Ile641, Val616)

    • Potential LC3-interacting regions

    • Membrane targeting domains

  • Compare effects of these mutants on autophagy to distinguish functional domains

• Rescue Experiments:

  • After OSBPL7 knockdown, attempt rescue with:

    • Wild-type OSBPL7

    • OSBPL7 mutants

    • Other OSBPL family members

  • Selective rescue indicates pathway specificity

• Acute Inactivation:

  • Employ degron-based systems for rapid OSBPL7 protein depletion

  • Monitor immediate autophagy consequences before compensatory mechanisms emerge

• Direct Interaction Verification:

  • Use techniques with increasing stringency to confirm interactions:

    • Proximity labeling (BioID/APEX)

    • Co-immunoprecipitation with biotin-conjugated antibodies

    • In vitro binding assays with purified components

    • FRET/BRET analysis for in vivo interactions

Research indicates that OSBPL7 interacts with the unlipidated form of LC3B (LC3B-I) , suggesting a pre-autophagosomal role. When designing experiments, researchers should consider whether OSBPL7's effects stem from this direct interaction or from its lipid transfer activity altering membrane composition required for autophagosome formation.

What emerging techniques can enhance the utility of biotin-conjugated anti-OSBPL7 antibodies in studying membrane contact sites?

Emerging techniques offer new possibilities for investigating OSBPL7 at membrane contact sites:

• Proximity Labeling with Biotin-Conjugated Antibodies:

  • Combine biotin-conjugated anti-OSBPL7 with peroxidase-mediated proximity labeling

  • This approach can identify proteins within nanometer-scale proximity of OSBPL7 at membrane contact sites

  • Allows mapping of the OSBPL7 interactome specifically at ER-autophagosome contact sites

• Super-Resolution Imaging Enhancement:

  • Deploy biotin-conjugated antibodies with small fluorophore-streptavidin conjugates for STORM/PALM microscopy

  • Achieve 10-20 nm resolution of OSBPL7 localization relative to membrane contact site markers

  • Combine with expansion microscopy techniques for enhanced spatial resolution

• Live-Cell Adaptation Strategies:

  • Develop cell-permeable biotin-conjugated nanobodies against OSBPL7

  • Enable dynamic tracking of endogenous OSBPL7 during autophagosome formation

  • Pair with optogenetic tools to manipulate OSBPL7 function with spatial and temporal precision

• Correlative Light and Electron Microscopy (CLEM):

  • Use biotin-conjugated antibodies with gold-streptavidin for ultrastructural localization

  • Correlate fluorescence patterns with electron microscopy to visualize OSBPL7 at membrane contact sites with nanometer precision

  • Apply cryo-electron tomography to preserve native membrane architecture

These approaches are particularly valuable for investigating OSBPL7's proposed role as a lipid transfer protein at membrane contact sites during autophagosome formation , potentially revealing how lipid composition changes facilitate membrane remodeling during autophagy.

How might biotin-conjugated anti-OSBPL7 antibodies contribute to drug discovery targeting OSBPL7-mediated pathways?

Biotin-conjugated anti-OSBPL7 antibodies can accelerate therapeutic development through several applications:

• High-Throughput Screening Platforms:

  • Develop ELISA-based assays using biotin-conjugated antibodies to screen compound libraries

  • Identify molecules that:

    • Modulate OSBPL7 protein levels or stability

    • Alter OSBPL7 interaction with binding partners

    • Change OSBPL7 subcellular distribution

  • Adapt to automated 384-well formats for large-scale screening

• Target Engagement Verification:

  • Use cellular thermal shift assays (CETSA) with biotin-conjugated antibodies to confirm direct binding of compounds to OSBPL7

  • Verify compound specificity across OSBPL family members

  • Assess on-target activity in complex cellular environments

• Pharmacodynamic Biomarker Development:

  • Establish assays to monitor OSBPL7 pathway modulation in response to therapeutic intervention

  • Develop multiplexed detection of OSBPL7 and downstream effectors (e.g., ABCA1, cholesterol efflux markers)

  • Create methods suitable for ex vivo analysis of patient samples in clinical trials

• Drug Resistance Mechanisms:

  • Investigate adaptations in OSBPL7 expression, localization, or interaction profiles associated with treatment resistance

  • Screen for combination approaches that prevent resistance emergence

Recent research identified 5-arylnicotinamide compounds that target OSBPL7's sterol binding pocket, increasing ABCA1-dependent cholesterol efflux and showing therapeutic potential in kidney disease models . Biotin-conjugated antibodies can facilitate further refinement of these compounds and identification of new therapeutic candidates by providing highly sensitive detection methods for pathway modulation.

What are the methodological considerations for investigating OSBPL7's role in diseases beyond kidney disorders?

OSBPL7's involvement in fundamental cellular processes suggests broader disease relevance requiring specific methodological approaches:

• Neurodegenerative Disorders:

  • Apply biotin-conjugated anti-OSBPL7 antibodies to brain tissue sections with specialized protocols:

    • Extended antigen retrieval for formalin-fixed tissues

    • Lipofuscin autofluorescence quenching

    • Co-staining with markers of protein aggregates (Aβ, tau, α-synuclein)

  • Investigate OSBPL7's relationship with neuronal autophagy defects common in neurodegenerative conditions

  • Examine OSBPL7 expression in microglia and astrocytes during neuroinflammation

• Metabolic Disorders:

  • Develop tissue-specific protocols for adipose, liver, and muscle samples:

    • Optimize fixation to preserve lipid structures

    • Co-localization with lipid droplet markers

    • Quantitative assessment of OSBPL7 expression relative to metabolic state

  • Investigate OSBPL7's potential role in hepatic lipid metabolism and non-alcoholic fatty liver disease

• Cancer Biology:

  • Create tissue microarray screening approaches:

    • High-throughput IHC with biotin-conjugated antibodies

    • Digital pathology quantification of expression patterns

    • Correlation with patient outcomes and treatment response

  • Examine OSBPL7's potential influence on autophagy-dependent survival mechanisms in cancer cells

• Immune Disorders:

  • Develop flow cytometry applications:

    • Intracellular staining protocols for OSBPL7

    • Multi-parameter analysis with immune cell markers

    • Sorting strategies for OSBPL7-high/low populations

  • Investigate OSBPL7's role in selective autophagy during pathogen clearance

OSBPL7's documented roles in both cholesterol metabolism and selective autophagy pathways suggest it may represent an unexplored node connecting lipid homeostasis with quality control mechanisms across multiple disease contexts. When designing studies beyond kidney disorders, researchers should consider tissue-specific expression patterns and potential compensatory mechanisms from other OSBPL family members.