OSBP Antibody, FITC conjugated

What is the OSBP protein family and what cellular functions do they serve?

The OSBP (Oxysterol Binding Protein) family consists of closely related proteins characterized by two major structural elements: a highly conserved C-terminal oxysterol binding domain and, in most cases, an N-terminal pleckstrin homology (PH) domain. These proteins are involved in multiple cellular functions including lipid metabolism, vesicle transport, and cell signaling pathways . The OSBP family includes multiple related proteins (OSBPLs) with varying tissue distributions and subcellular localizations. For example, OSBPL3 is expressed in kidney and lymphatic tissues as well as blood leukocytes including B-cells, T-cells, and macrophages . When selecting an antibody for your research, understanding the specific OSBP family member's distribution is crucial for appropriate experimental design.

What are the key differences between OSBP and OSBPL antibodies?

While both target members of the oxysterol binding protein family, the specific proteins they recognize have distinct cellular distributions and functions. OSBP primarily refers to the original oxysterol binding protein (full name: oxysterol binding protein), which has a molecular weight of approximately 89 kDa . OSBPLs (OSBP-Like proteins) are related family members with varying functions. For instance, OSBPL7 (ORP-7) is associated with autophagosome, cytosol, perinuclear endoplasmic reticulum, and plasma membrane locations, and has roles in cholesterol binding, sterol transport, bile acid biosynthesis, cellular cholesterol response, proteasomal protein catabolism, and autophagy regulation . When designing experiments, ensure you're targeting the specific family member relevant to your research question.

What applications are FITC-conjugated OSBP antibodies suitable for?

FITC-conjugated OSBP antibodies are particularly valuable for direct visualization techniques. Based on available data, these antibodies are primarily used in:

For OSBPL7 antibody with FITC conjugation, ELISA applications have been specifically validated . When designing experiments utilizing fluorescence detection, these conjugated antibodies eliminate the need for secondary antibody incubation steps, simplifying protocols and potentially reducing background signal.

How can OSBP antibodies be utilized in cancer research investigations?

Research demonstrates that OSBP family proteins may play significant roles in cancer progression. For example, OSBPL3 has been shown to be upregulated in colorectal cancer (CRC) tissues compared to normal tissues. High expression of OSBPL3 correlates with poor differentiation, advanced TNM stage, and poor prognosis in CRC patients . In experimental settings, OSBPL3 has been demonstrated to promote proliferation, invasion, and metastasis of CRC through activation of the RAS signaling pathway .

When investigating OSBP family proteins in cancer:

• Consider the specific family member expression in your cancer model

• Correlate expression levels with clinical parameters

• Examine potential involvement in relevant signaling pathways

• Use both in vitro and in vivo models for comprehensive analysis

Kaplan-Meier survival analyses from published CRC datasets have shown that higher OSBPL3 expression significantly correlates with poorer survival of patients (log-rank, p < 0.05) .

What considerations should be made when using FITC-conjugated antibodies in multicolor immunofluorescence experiments?

When designing multicolor immunofluorescence experiments with FITC-conjugated OSBP antibodies, several technical considerations are essential:

• Spectral Overlap Management: FITC has an emission spectrum that may overlap with other commonly used fluorophores. Consider the following table when selecting additional fluorescent markers:

• Sequential Imaging: For confocal microscopy, sequential rather than simultaneous acquisition may be necessary to avoid bleed-through.

• Fixation Considerations: FITC fluorescence is pH-sensitive, so buffer selection during fixation and permeabilization is critical. Protocols typically use 4′,6-diamidino-2-phenylindole (DAPI) for counterstaining nuclei when working with FITC-conjugated antibodies .

How do expression patterns of OSBP family members correlate with disease progression?

Analysis of OSBP family member expression in relation to disease states reveals important patterns. For OSBPL3, expression was found to be positively correlated with the stage of colorectal cancer . Immunohistochemistry staining showed that OSBPL3 displayed cytoplasmic or cytoplasmic/membrane localization in CRC cells, with upregulation in cancer tissues compared to matched adjacent normal tissues, and even higher expression in those with distant metastasis .

Statistical analysis of 133 CRC patient samples showed significant correlations between high OSBPL3 expression and:

• Poor differentiation

• Advanced TNM stage

• Advanced Dukes stage

When investigating OSBP family proteins in disease progression, researchers should consider:

• Subcellular localization changes during disease progression

• Correlation with established clinical staging parameters

• Potential as prognostic biomarkers

What are the optimal dilution ratios for different experimental applications of OSBP antibodies?

Optimal dilution ratios vary significantly by application. For OSBP antibody (11096-1-AP), the following dilutions are recommended:

For every new experimental system, it is strongly recommended to perform antibody titration to determine the optimal concentration that provides specific signal with minimal background .

What fixation and permeabilization protocols are recommended for immunofluorescence with FITC-conjugated OSBP antibodies?

For optimal results with FITC-conjugated antibodies in immunofluorescence applications, consider the following protocol framework based on published methodologies:

• Cell Preparation: Seed cells (5 × 10^4/well) on coverslips and allow growth for 48 hours

• Fixation Options:

  • 4% paraformaldehyde (15 minutes at room temperature)

  • 100% methanol (10 minutes at -20°C) for membrane proteins

• Permeabilization (if using paraformaldehyde): 0.1-0.5% Triton X-100 in PBS (5-10 minutes)

• Blocking: 1-5% BSA or normal serum in PBS (30-60 minutes)

• Antibody Incubation: Apply FITC-conjugated antibody at optimized dilution (typically 1:20-1:200) in blocking buffer

• Counterstaining: DAPI for nuclear visualization

• Mounting: Anti-fade mounting medium to minimize photobleaching

Remember that FITC is sensitive to pH; maintaining a slightly alkaline environment (pH 7.4-8.0) will help preserve fluorescence intensity.

What controls should be included when using OSBP antibodies in research applications?

Rigorous control inclusion is essential for reliable interpretation of results with OSBP antibodies:

• Positive Controls: Include samples known to express the target protein. For OSBP (11096-1-AP), validated positive controls include:

  • HEK-293 cells

  • HeLa cells

  • Rat and mouse kidney tissue

  • Rat and mouse brain tissue

• Negative Controls:

  • Isotype control (same host species IgG)

  • Samples with confirmed absence of target protein

  • For FITC-conjugated antibodies, include an unstained sample to account for autofluorescence

• Knockdown/Knockout Validation: For definitive specificity confirmation, include samples with gene knockdown or knockout. Published literature has validated OSBP antibodies in KD/KO systems in multiple publications .

• Absorption Controls: Pre-incubation of antibody with immunizing peptide should abolish specific staining.

How can background signal be reduced when using FITC-conjugated OSBP antibodies?

Background signal can significantly impact the interpretability of results. Consider these strategies for optimization:

• Antibody Concentration: Titrate to determine the minimum concentration that provides specific signal. Start with recommended dilutions (1:20-1:200 for IF applications) and adjust as needed.

• Blocking Optimization:

  • Increase blocking time (1-2 hours)

  • Try different blocking agents (BSA, normal serum, commercial blockers)

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

• Buffer Considerations:

  • Ensure slightly alkaline pH (7.4-8.0) to maintain FITC fluorescence

  • Add 0.01-0.05% Tween-20 to wash buffers

  • Consider PBS with 0.02% sodium azide and 50% glycerol pH 7.3 for storage

• Sample Preparation:

  • Fresh fixation (avoid over-fixation)

  • Adequate permeabilization for intracellular targets

  • Proper storage of antibody (aliquoting is recommended for long-term storage)

What approaches can resolve discrepancies between OSBP antibody detection methods?

When facing inconsistent results between different detection methods (e.g., WB vs. IF), consider:

• Epitope Accessibility: Different detection methods expose different protein epitopes. For OSBP family proteins, the conformation in fixed cells (IF) may differ from denatured proteins (WB).

• Antibody Validation: Confirm antibody specificity using multiple methods. For example, OSBP antibody (11096-1-AP) has been validated in:

  • WB across multiple cell lines and tissues

  • IP in HeLa cells

  • IHC in human ovary tumor tissue

  • IF/ICC in HeLa cells

• Protocol Adjustments:

  • For IHC: Try different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • For WB: Modify transfer conditions for high molecular weight proteins (89 kDa for OSBP)

  • For IF: Adjust fixation and permeabilization parameters

• Isoform Specificity: Consider whether your antibody recognizes all or specific isoforms of your target OSBP family protein.

How should researchers approach co-localization studies using FITC-conjugated OSBP antibodies?

Co-localization studies require special considerations:

• Microscopy Settings:

  • Use sequential scanning in confocal microscopy to prevent bleed-through

  • Apply appropriate negative controls for each channel

  • Maintain consistent PMT/gain settings between samples

• Quantitative Analysis:

  • Calculate Pearson's or Manders' coefficients for objective assessment

  • Use multiple fields of view (minimum 5-10 per condition)

  • Include biological replicates (minimum n=3)

• Combining with Other Markers:

  • For studying OSBPL7 localization, consider co-staining with markers for:

    • Autophagosome (LC3)

    • Endoplasmic reticulum (Calnexin)

    • Plasma membrane (WGA or Na+/K+ ATPase)

• Technical Validation:

  • Perform reverse staining (swap fluorophores between antibodies)

  • Include single-stained controls for spillover correction