OSBPL7 Antibody, HRP conjugated

What is OSBPL7 and why is it significant in research applications?

OSBPL7 is a member of the oxysterol binding protein family that plays a crucial role in cellular cholesterol homeostasis and lipid metabolism. Research significance stems from its involvement in ABCA1-dependent cholesterol efflux pathways and implications in kidney disease, cardiovascular disorders, and inflammatory conditions. OSBPL7 contains a highly conserved oxysterol regulatory domain (ORD) at its carboxy-terminus that forms a sterol binding pocket, allowing it to interact with various lipid molecules . Studies have demonstrated that compounds targeting OSBPL7 can upregulate ABCA1-dependent cholesterol efflux, representing a potential therapeutic strategy for treating renal diseases and other disorders involving cellular cholesterol dysregulation .

How does OSBPL7 function at the molecular level in cellular systems?

OSBPL7 functions primarily through its sterol binding pocket, which accommodates cholesterol and other lipid molecules. Homology modeling has revealed that key amino acid residues like Lys636, Ile641, Val616, and Val618 line this binding pocket and are essential for its functionality . The protein interacts with multiple cellular pathways, particularly those involving lipid transport and metabolism. OSBPL7 has been localized to both the endoplasmic reticulum and plasma membrane, suggesting a role in intracellular lipid trafficking . Additionally, OSBPL7 interacts with central metabolic regulators such as AKT1, indicating its involvement in broader metabolic signaling networks . When inhibited or silenced, OSBPL7 leads to increased ABCA1-dependent cholesterol efflux to apolipoprotein A1, altered triacylglycerol levels, and changes in membrane fluidity .

What detection methods are available for OSBPL7 in biological samples?

Multiple detection methods exist for OSBPL7 quantification and localization in biological samples:

• ELISA Assays: Enzyme-linked immunosorbent assays are available specifically designed for OSBPL7 detection in human serum, plasma, cell culture supernatant, and other biological samples. Commercial kits have been validated particularly for citrated/EDTA plasma samples .

• Western Blotting: This technique allows for protein size confirmation and relative quantification. When using HRP-conjugated antibodies, direct detection is possible without secondary antibodies, simplifying the workflow and potentially reducing background signal .

• Immunoprecipitation: This approach has been successfully employed to study OSBPL7 interactions with other proteins, as demonstrated in studies examining potential associations between OSBPL7 and ABCA1 .

• qPCR: For mRNA-level detection, quantitative PCR has been utilized to measure OSBPL7 expression changes in response to various treatments or experimental conditions .

What are the key advantages of using HRP-conjugated OSBPL7 antibodies?

HRP-conjugated OSBPL7 antibodies offer several methodological advantages for researchers:

• Streamlined Protocols: By eliminating the need for secondary antibody incubation steps, these conjugated antibodies reduce experimental time and potential sources of variability.

• Reduced Background Noise: Direct detection systems often demonstrate lower background levels compared to indirect detection methods, particularly in complex biological samples.

• Enhanced Sensitivity: HRP enzyme amplification provides excellent signal generation capabilities, allowing for detection of low-abundance OSBPL7 protein in various sample types.

• Multiplexing Capability: When different detection enzymes are used with other antibodies, these conjugated antibodies facilitate simultaneous detection of multiple targets in the same sample.

• Compatibility: These antibodies are compatible with standard detection substrates like TMB, ECL reagents, and colorimetric substrates, offering versatility across detection platforms .

What considerations are important when designing OSBPL7 detection experiments?

When designing experiments to detect or quantify OSBPL7, researchers should consider:

• Sample Preparation: Different biological samples require specific preparation techniques. For membrane-associated OSBPL7, appropriate detergent extraction is essential. OSBPL7 has been successfully detected in cell lysates using standard lysis buffers containing protease inhibitors .

• Expression Levels: OSBPL7 expression varies across tissues and cell types. Podocytes and endothelial cells have been successfully used to study OSBPL7 function .

• Specificity Controls: Include OSBPL7 knockdown or overexpression controls to validate antibody specificity. Researchers have employed siRNA knockdown of OSBPL7 (achieving approximately 60% reduction in mRNA) to confirm antibody specificity .

• Cross-Reactivity: Consider potential cross-reactivity with other OSBP family members, particularly those with high sequence homology to OSBPL7. The OSBP family contains several members with similar oxysterol binding domains .

• Signal Amplification: For low-abundance samples, consider signal amplification strategies such as tyramide signal amplification when using HRP-conjugated antibodies.

How can I optimize Western blotting protocols for OSBPL7 detection?

For optimal Western blot detection of OSBPL7 using HRP-conjugated antibodies:

• Sample Preparation: Use cell lysis buffers containing 1% Triton X-100 or RIPA buffer with protease inhibitors to effectively extract OSBPL7 from membrane structures.

• Protein Loading: Load 20-50 μg of total protein per lane, depending on OSBPL7 expression levels in your sample type. Adjust based on preliminary results.

• Gel Percentage: Use 8-10% polyacrylamide gels for optimal resolution of OSBPL7 (molecular weight approximately 95-100 kDa).

• Transfer Conditions: For efficient transfer of high molecular weight OSBPL7, perform wet transfer at 30V overnight at 4°C or 100V for 1 hour with cooling.

• Blocking: Block membranes with 5% non-fat milk or 3% BSA in TBST for 1 hour at room temperature to minimize non-specific binding.

• Antibody Dilution: Typically start with 1:1000 dilution for HRP-conjugated OSBPL7 antibodies, then optimize based on signal-to-noise ratio in your specific system.

• Incubation Time: Incubate with primary antibody for 2 hours at room temperature or overnight at 4°C for optimal results.

• Positive Controls: Include samples from cells overexpressing OSBPL7, as validated with approximately 16-fold overexpression compared to endogenous levels .

What strategies should be employed for OSBPL7 co-immunoprecipitation studies?

When conducting co-immunoprecipitation studies involving OSBPL7:

• Crosslinking Considerations: Consider using mild crosslinking agents (0.5-1% formaldehyde) to stabilize transient protein-protein interactions. UV crosslinking has been successfully employed in OSBPL7 interaction studies .

• Lysis Conditions: Use gentle lysis buffers (containing 0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions while effectively solubilizing membrane-associated OSBPL7.

• Antibody Selection: Choose antibodies with epitopes that don't interfere with potential protein interaction sites. For tagged proteins, use anti-tag antibodies (V5, FLAG) as demonstrated in previous OSBPL7-ABCA1 interaction studies .

• Bead Selection: Magnetic beads often provide better recovery and less non-specific binding than agarose beads. Anti-FLAG beads have been successfully used for immunoprecipitation of ABCA1-FLAG in studies investigating OSBPL7 interactions .

• Wash Stringency: Balance between removing non-specific interactions and preserving genuine interactions by optimizing salt concentration (150-300 mM NaCl) in wash buffers.

• Controls: Include IgG controls, input samples, and reciprocal IPs (pulling down with anti-OSBPL7 and blotting for suspected interactors, and vice versa) to validate interactions.

• Validation Techniques: Confirm interactions using alternative methods such as proximity ligation assays or FRET analysis. Previous studies utilized both co-immunoprecipitation and chemical biology approaches with photoactivatable compounds to identify OSBPL7 interactions .

How can OSBPL7 antibodies be utilized in studying cholesterol efflux mechanisms?

OSBPL7 antibodies can be instrumental in elucidating cholesterol efflux mechanisms through the following approaches:

• Proximity Studies: Use OSBPL7 antibodies in conjunction with ABCA1 antibodies to determine co-localization patterns at the plasma membrane using confocal microscopy.

• Functional Assays: Monitor OSBPL7 expression and localization changes during cholesterol efflux assays using antibody detection methods. Studies have demonstrated that OSBPL7 inhibition increases ABCA1-dependent cholesterol efflux to apolipoprotein A1 .

• Pathway Validation: Use OSBPL7 antibodies to confirm protein expression changes following siRNA silencing or pharmacological inhibition in cholesterol efflux studies. Approximately 60% reduction in OSBPL7 mRNA levels via siRNA has been shown to increase cholesterol efflux by approximately 50% .

• Target Engagement Validation: Use OSBPL7 antibodies to confirm binding of 5-arylnicotinamide compounds to OSBPL7 in cellular systems as part of target engagement studies .

• ABCG1 Pathway Investigation: Examine the relationship between OSBPL7 and ABCG1-mediated cholesterol efflux, as ORP7 inhibition has been shown to decrease ABCG1-mediated cholesterol efflux in endothelial cells .

What methods are effective for studying OSBPL7 in kidney disease models?

For investigating OSBPL7 in kidney disease models:

• Immunohistochemistry Protocol:

  • Use 4% paraformaldehyde-fixed kidney sections (5-7 μm thickness)

  • Perform antigen retrieval using citrate buffer (pH 6.0)

  • Block with 5% normal serum and 0.1% Triton X-100

  • Apply HRP-conjugated OSBPL7 antibody (1:100-1:500 dilution)

  • Develop with DAB substrate and counterstain with hematoxylin

  • Include podocyte markers (nephrin, podocin) for co-localization studies

• Disease Model Selection: OSBPL7-targeting compounds have shown efficacy in both Adriamycin-induced nephropathy and Alport Syndrome models, making these appropriate systems for studying OSBPL7 function in kidney disease .

• Functional Assessments: Combine OSBPL7 detection with functional assays such as proteinuria measurements, podocyte morphology assessment, and renal fibrosis quantification to correlate OSBPL7 levels with disease progression .

• Cell-Type Specific Analysis: Use OSBPL7 antibodies with cell-type specific markers to determine expression patterns across different kidney cell populations, particularly focusing on podocytes where OSBPL7 function has been well-characterized .

How can I troubleshoot weak signals when using OSBPL7 antibodies?

When encountering weak signals with OSBPL7 antibodies:

• Antibody Concentration: Increase primary antibody concentration in incremental steps (e.g., from 1:1000 to 1:500, 1:250) while monitoring background levels.

• Incubation Conditions: Extend primary antibody incubation time to overnight at 4°C to enhance binding efficiency without increasing background.

• Signal Amplification: For HRP-conjugated antibodies, use enhanced chemiluminescence (ECL) substrates with longer signal duration, or consider tyramide signal amplification for immunohistochemistry applications.

• Sample Preparation: Ensure complete solubilization of membrane-associated OSBPL7 by adjusting detergent concentrations or trying different extraction buffers.

• Protein Degradation: Add additional protease inhibitors to prevent OSBPL7 degradation during sample preparation, particularly when working with tissue samples.

• Epitope Accessibility: Consider mild denaturation methods or alternative antigen retrieval protocols to improve epitope accessibility in fixed samples.

• HRP Activity: Verify HRP activity of the conjugated antibody using a direct enzyme activity assay; if reduced, consider newer antibody lots or different storage conditions.

What approaches help differentiate between OSBPL7 and other OSBP family members?

To effectively distinguish OSBPL7 from other OSBP family members:

• Epitope Selection: Choose antibodies raised against unique regions of OSBPL7 outside the conserved ORD domain shared with other family members.

• Validation in Knockout/Knockdown Systems: Validate antibody specificity using OSBPL7 siRNA knockdown or CRISPR knockout cells, confirming signal reduction that corresponds with approximately 60-80% knockdown efficiency .

• Competition Assays: Perform peptide competition assays using the immunizing peptide to confirm antibody specificity.

• Expression Pattern Analysis: Compare detected patterns with known tissue-specific expression profiles of OSBPL7 versus other family members.

• Co-staining Experiments: Perform co-staining with antibodies against multiple OSBP family members to identify differential localization patterns.

• Western Blot Size Discrimination: Use high-resolution SDS-PAGE to separate OSBPL7 (approximately 95-100 kDa) from other OSBP family members with different molecular weights.

How are OSBPL7 antibodies being used in inflammatory and metabolic disease research?

OSBPL7 antibodies are finding increasing applications in inflammatory and metabolic disease research:

• Transcriptional Regulation Studies: Monitor OSBPL7 protein levels in response to inflammatory stimuli, as OSBPL7 inhibition has been associated with upregulation of proinflammatory genes .

• Lipid Metabolism Analysis: Use OSBPL7 antibodies to investigate protein expression changes in conditions of altered lipid metabolism, particularly focusing on ceramides and lysophosphatidylcholines which increase upon OSBPL7 inhibition .

• Angiogenesis Research: Employ OSBPL7 antibodies to study protein localization during angiogenic processes, as OSBPL7 inhibition results in reduced angiogenic capacity in endothelial cells .

• AKT1 Interaction Studies: Investigate OSBPL7-AKT1 interactions using co-immunoprecipitation with OSBPL7 antibodies, building on interactomic findings that revealed this potentially important metabolic regulation pathway .

• Macroautophagy Research: Examine OSBPL7's role in macroautophagy through co-staining with LC3B, as OSBPL7 has been shown to bind to unlipidated LC3B and play a role in the macroautophagy pathway .

What methodological considerations apply when using OSBPL7 antibodies in high-throughput screening?

For high-throughput screening applications using OSBPL7 antibodies:

• Assay Miniaturization: Optimize antibody concentration and detection methods for 384- or 1536-well formats, typically starting with 2-5 fold higher antibody concentrations than recommended for standard formats.

• Automation Compatibility: Ensure HRP-conjugated antibody formulations are compatible with liquid handling systems by testing for aggregation or adhesion issues.

• Signal Window Optimization: Establish robust positive and negative controls to achieve Z' factors >0.5, essential for distinguishing true hits in screening campaigns.

• Readout Selection: For HRP-conjugated antibodies, select stable luminescent substrates for plate-reading applications or fluorescent substrates for imaging-based high-content screening.

• Assay Validation: Validate high-throughput assays using known OSBPL7 modulators such as the 5-arylnicotinamide compounds that have demonstrated activity in previous studies .

• Multiplexing Strategies: Consider multiplexed detection approaches that combine OSBPL7 detection with other pathway components (e.g., ABCA1) to increase information content per well.

What are the latest developments in using OSBPL7 antibodies for therapeutic target validation?

Recent developments in therapeutic target validation using OSBPL7 antibodies include:

• Target Engagement Studies: OSBPL7 antibodies have been employed to validate binding of 5-arylnicotinamide compounds to OSBPL7 in photoaffinity labeling and competition experiments, confirming the compound-target interaction central to their therapeutic effects .

• Disease Model Validation: In kidney disease models (Adriamycin-induced nephropathy and Alport Syndrome), OSBPL7 antibodies have helped confirm target presence and modulation in response to therapeutic interventions .

• Pathway Analysis: OSBPL7 antibodies are being used to elucidate the mechanisms connecting OSBPL7 inhibition to increased ABCA1-dependent cholesterol efflux, a potentially therapeutic effect for kidney and cardiovascular diseases .

• Biomarker Development: Researchers are investigating OSBPL7 detection in biological fluids using ELISA methods as potential biomarkers for diseases associated with altered cholesterol homeostasis .

• Resistance Mechanism Studies: In therapeutic approaches targeting OSBPL7, antibodies help monitor potential compensatory changes in related pathways or family members that could contribute to treatment resistance.

How should researchers interpret changes in OSBPL7 expression across different experimental conditions?

When interpreting OSBPL7 expression changes:

• Context-Dependent Effects: Consider that OSBPL7 inhibition produces different effects depending on cell type—increasing ABCA1-dependent cholesterol efflux in podocytes but potentially reducing ABCG1-mediated efflux in endothelial cells .

• Quantification Methods: Use appropriate normalization methods for Western blots (housekeeping proteins) and qPCR (reference genes), as demonstrated in studies showing approximately 1.36-fold increase in ABCA1 expression after OSBPL7 inhibition .

• Temporal Considerations: Account for time-dependent changes in OSBPL7 expression and downstream effects, as pathway alterations may require sufficient time to manifest.

• Threshold Effects: Consider that biological effects may require a minimum threshold of OSBPL7 modulation—siRNA knockdown of approximately 60% was sufficient to stimulate a 50% increase in cholesterol efflux .

• Integrated Analysis: Combine protein-level changes (Western blot/ELISA) with functional readouts (cholesterol efflux, angiogenesis assays) for comprehensive interpretation of OSBPL7's role .

What statistical approaches are most appropriate for analyzing OSBPL7 expression data?

For rigorous statistical analysis of OSBPL7 expression data:

• Sample Size Determination: Power analysis should be performed to determine appropriate sample sizes, typically requiring at least n=3-5 biological replicates for preliminary studies and n=6-10 for definitive experiments.

• Normality Testing: Apply Shapiro-Wilk or D'Agostino-Pearson tests to determine if OSBPL7 expression data follows normal distribution.

• Parametric vs. Non-parametric Tests: For normally distributed data, use t-tests (two groups) or ANOVA (multiple groups); for non-normally distributed data, use Mann-Whitney (two groups) or Kruskal-Wallis (multiple groups) tests.

• Multiple Comparisons: Apply Bonferroni, Tukey, or Dunnett corrections when comparing multiple experimental conditions to control for family-wise error rate.

• Correlation Analysis: When examining relationships between OSBPL7 levels and functional outcomes (e.g., cholesterol efflux), use Pearson (parametric) or Spearman (non-parametric) correlation analyses.

• Visualization Methods: Present data using box plots or violin plots rather than simple bar graphs to better represent data distribution and variability.

• Effect Size Reporting: Report Cohen's d or similar effect size metrics alongside p-values to indicate biological significance beyond statistical significance.