HSP15.7 Antibody

Basic Research Questions

How do I validate antibody specificity for Hsp70 in immunoprecipitation (IP) experiments?

• Perform parallel negative controls using:

  • Isotype-matched control antibodies (e.g., non-specific mouse IgG)

  • Antigen-blocking experiments: Pre-incubate the antibody with recombinant Hsp70 protein to confirm signal loss .

  • Knockdown/knockout validation: Compare results in Hsp70-depleted cell lines vs. wild-type .

What methodologies optimize Hsp70 detection in formaldehyde-fixed cells?

• Use antigen retrieval with 10 mM sodium citrate (pH 6.0) microwaved for 8–15 minutes .

• Block with 10% human serum/PBS to reduce background .

• Validate with secondary antibody-only controls to rule out non-specific binding (Figure 3 in ).

How to select antibodies for Hsp70 isoforms across species?

• Prioritize clones with cross-reactivity validation (e.g., ab5442 works for human, mouse, and yeast) .

• Confirm using Western blot against recombinant proteins from target species.

Advanced Research Questions

How to design experiments investigating Hsp15's role in ribosomal rescue (e.g., in E. coli)?

• Key steps:

  • Induce heat shock at 42°C for 15 min to accumulate 50S ribosomal subunits .

  • Use FLAG-tagged Hsp15 for co-immunoprecipitation with anti-FLAG antibodies .

  • Analyze complexes via cryo-EM to resolve Hsp15-50S-peptidyl-tRNA structures (3.0 Å resolution achieved in ).

• Critical controls:

  • Compare with non-heat-shocked samples

  • Include tRNA-free ribosomal fractions

How to resolve contradictions in anti-Hsp70 autoantibody data across biological fluids?

• Validate using orthogonal methods (e.g., ELISA + Western blot) .

What multi-modal approaches confirm Hsp70’s role in heat-induced pathologies?

• Combine:

  • Clinical data: Anti-Hsp71 antibody titers in heat-stroke patients (1:80 in severe cases vs. 1:20 controls) .

  • Cellular assays: Hsp70 overexpression in heat-shocked leukocytes .

  • Longitudinal analysis: Track antibody titer changes during recovery (↓50% post-treatment in ).

Methodological Guidance Table

Key Research Findings

• Anti-Hsp70 autoantibodies exhibit fluid-specific isotype distributions:

  • Saliva: IgG (2.1 ± 0.3 OD450) and IgA (1.8 ± 0.2 OD450)

  • Serum: IgG only (3.4 ± 0.4 OD450)

• Hsp15 stabilizes peptidyl-tRNA in a 50S ribosomal P-site orientation distinct from bacterial RqcP .

• Anti-Hsp71 titers correlate with heat-stroke severity (ROC AUC = 0.82, p < 0.01) .