ORP2B Antibody

Here’s a structured, research-focused FAQ collection for "ORP2B Antibody" based on academic literature and experimental methodologies:

How to validate ORP2B antibody specificity in plant and mammalian systems?

Methodology:

• Perform siRNA/shRNA knockdown of OSBPL2 (human ortholog of ORP2B) in cell lines (e.g., HEK293T) and compare Western blot (WB) signals pre/post knockdown .

• Use CRISPR-Cas9 knockout lines (e.g., Arabidopsis orp2a-1 mutants) to confirm antibody specificity in plant systems .

• Validate cross-reactivity using recombinant ORP2B protein and peptide-blocking assays (e.g., immunogen: OSBPL2 aa 250–C terminus) .

Data Table:

What cellular processes should ORP2B antibody target for functional studies?

Methodology:

• Focus on lipid transport roles:

  • Co-localization with ER-PM contact sites (EPCS) using confocal microscopy (e.g., VAP27-1 interaction in Arabidopsis) .

  • Detect changes in phosphatidylinositol-4,5-bisphosphate (PIP2) levels via lipid-binding assays .

• Study glucose signaling pathways by monitoring AtRGS1 degradation kinetics in plant mutants .

Key Applications:

• Immunohistochemistry (IHC-P) for subcellular localization .

• Co-IP/MS to identify interactors (e.g., VAP27-1, AtRGS1) .

How to resolve contradictory data in ORP2B-linked signaling pathways?

Case Study:
In Arabidopsis, ORP2A (ortholog of ORP2B) shows dual roles in G protein signaling and lipid transport. Contradictions arise from:

• Tissue-specific splice variants (100 kDa vs. 70 kDa isoforms) .

• Context-dependent interactions (e.g., AtRGS1 in sugar response vs. VAP27-1 in EPCS) .

Resolution Strategy:

• Use isoform-specific antibodies (e.g., N-terminal vs. C-terminal epitopes).

• Conduct transcriptome-wide KEGG analysis to identify pathway overlaps (e.g., 40% shared DEGs between orp2a-1 and agb1-2 mutants) .

Data Table: Mutant Phenotypes in Arabidopsis

How to design experiments distinguishing ORP2B’s lipid transport vs. signaling roles?

Experimental Framework:

• Lipid Binding:

  • Use PIP strips with recombinant ORP2B (prioritize phosphatidic acid and PI3P) .

  • Quantify membrane cholesterol via filipin staining in ORP2B-KO cells .

• Signaling Modulation:

  • Measure GTPase activity in G protein-coupled pathways (e.g., glucose response in Arabidopsis) .

  • Track AtRGS1 endocytosis via live-cell imaging with pH-sensitive tags .

Critical Controls:

• Include OSBPL2-rescue constructs to confirm phenotype reversibility.

• Use orthogonal methods (e.g., SPR for binding affinity vs. cellular assays) .

What computational tools predict ORP2B-antibody interactions for epitope mapping?

Methodology:

• AI-Based Design: Train deep learning models on OSBP family structures (e.g., AlphaFold-predicted ORP2B) to identify conformational epitopes .

• MD Simulations: Simulate antibody-antigen binding under lipid-rich conditions to assess sterol competition .

Validation Pipeline:

Methodological Notes

• Antibody Storage: Aliquot in 50% glycerol/PBS at -20°C to prevent lipid interference .

• Avoid Pitfalls:

  • Pre-adsorb antibodies with plant cell lysates to reduce non-specific binding .

  • Use gradient SDS-PAGE (8–12%) to resolve ORP2B isoforms .