VPS51 Antibody

Here’s a structured collection of research-focused FAQs for VPS51 antibodies, incorporating methodological guidance and data from peer-reviewed sources:

What experimental applications are validated for VPS51 antibodies, and how should researchers verify specificity?

VPS51 antibodies are primarily validated for:

• Western blot (WB): Detect endogenous VPS51 (~86 kDa) in human/mouse lysates .

• Immunohistochemistry (IHC): Localize VPS51 in Golgi-associated structures .

• Antibody competition assays: Confirm specificity using recombinant VPS51 antigens (e.g., His6-ABP-tagged antigens in PBS/urea buffer) .

Methodological validation steps:

• Positive controls: Use lysates from cell lines with confirmed VPS51 expression (e.g., HEK293T).

• Knockdown/knockout controls: Compare signal reduction in siRNA-treated or CRISPR-modified samples .

• Blocking peptide validation: Pre-incubate antibody with immunogen peptide (e.g., residues 620–670 for C-terminal antibodies) to assess signal loss .

How do I select the optimal VPS51 antibody for my experimental model?

Consider these parameters:

Example workflow:

• For WB in human cells: Use CAB15651 (rabbit polyclonal, validated in WB) .

• For IHC in mouse tissue: A46609 (rabbit polyclonal, 1.6 mg/mL, human/mouse cross-reactive) .

How can I resolve inconsistent VPS51 antibody signals across experimental replicates?

Common pitfalls and solutions:

What strategies are effective for multiplex detection of VPS51 with other trafficking proteins?

Recommended approach:

• Cross-validate antibody compatibility:

  • Use host species-matched antibodies (e.g., pair rabbit anti-VPS51 with mouse anti-VPS53).

  • Select fluorophores with non-overlapping spectra (e.g., Alexa Fluor 488/647) .

• Controls for retrograde transport studies:

  • Co-stain with TGN46 (trans-Golgi marker) and EEA1 (early endosome marker).

  • Quantify co-localization using Manders’ coefficients .

How can VPS51 antibodies be used to study disease-associated trafficking defects?

Case study: Neurodegeneration research

• Model system: Fibroblasts from pontocerebellar hypoplasia (PCH13) patients .

• Methods:

  • Compare VPS51 localization (IHC) and expression (WB) in patient vs. control cells .

  • Perform rescue experiments with WT VPS51 overexpression and monitor endosome-TGN trafficking .

• Critical controls: Include cells treated with Bafilomycin A1 (blocks endosomal acidification) to assess trafficking dependence on VPS51 .

Data Contradiction Analysis

Scenario: A study reports VPS51 at 86 kDa in WB, but your data show a 70 kDa band.
Resolution steps:

• Post-translational modifications: Treat lysates with PNGase F (removes N-glycans) or phosphatases .

• Isoform detection: Humans express 2 VPS51 isoforms; design isoform-specific primers for RT-PCR .

• Antibody mapping: Use truncation mutants to identify recognized epitopes (e.g., C-terminal vs. N-terminal antibodies) .