VPS9A Antibody

Basic Research Questions

1. Validating VPS9A Antibody Specificity in Arabidopsis thaliana
To confirm antibody specificity, perform immunoblotting using vps9a mutant lines (e.g., vps9a-2 with a truncated protein). Compare band sizes between wild-type (WT) and mutant tissues: WT samples should show a ~70-kDa band, while mutants exhibit a truncated ~30-kDa product . Include positive controls (e.g., recombinant VPS9a) and negative controls (mutant lysates). Cross-reactivity can be ruled out by testing unrelated Rab GTPases (e.g., RAB1 or RAB7) .

2. Confirming VPS9A’s Role as a Rab5 GEF
Use yeast two-hybrid assays with nucleotide-locked Rab5 variants:

• GDP-bound (S24N for ARA7/RHA1; S47N for ARA6) and nucleotide-free mutants interact with VPS9a .

• GTP-bound mutants (Q69L/Q93L) show no interaction .
  Supplement with pull-down assays using GST-tagged Rab5 preloaded with GDP/GTPγS to validate nucleotide-dependent binding .

3. Detecting Tissue-Specific VPS9A Expression
Combine immunoblotting with tissue-specific promoters (e.g., root vs. shoot). Published microarray data (e.g., Arabidopsis Expression Atlas) corroborate ubiquitous expression . For low-abundance tissues, use chemiluminescent detection with extended exposure times.

Advanced Experimental Design

4. Troubleshooting VPS9A Localization in Mutant Backgrounds
If endosomal localization is inconsistent:

• Test PUF2 co-expression: PUF2 recruits VPS9a to endosomes; puf2 mutants reduce VPS9a puncta by ~60% .

• Apply pharmacological agents:

  • Brefeldin A (BFA): Dissolves VPS9a-positive compartments .

  • Wortmannin (Wm): Induces endosomal dilation; measure diameter changes (WT: 1.98 µm vs. puf2: 1.60 µm) .

5. Resolving Contradictory Interaction Data
If Rab5-VPS9a binding results conflict:

• Verify nucleotide states: Use EDTA to strip nucleotides, then reload with GDP/GTPγS .

• Compare assay systems: Yeast two-hybrid may detect transient interactions, while pull-downs require stable binding (e.g., ARA6 interactions are transient and harder to capture) .

6. Analyzing VPS9A in Endosomal Fusion
Quantify Wm-induced endosomal dilation:

• Express fluorescent markers (e.g., ARA6-GFP and mRFP-ARA7).

• Measure diameter shifts post-Wm treatment (30% reduction in puf2 mutants) .

Key Experimental Data Tables

Table 1: VPS9a GEF Activity Toward Rab5 Isoforms

Table 2: Pharmacological Effects on VPS9a Localization

Methodological Notes

• Antibody Storage: Aliquot and store at -80°C; avoid freeze-thaw cycles to prevent degradation.

• Imaging: Use confocal microscopy with ≤1.5 µm optical slices for precise endosomal tracking .

• Quantitative Analysis: Measure ≥50 endosomes per condition for statistical rigor .