CLPC1 Antibody

Basic Research Questions

• How do I select CLPC1 antibodies for domain-specific studies in Mycobacterium tuberculosis?

  • Methodology:

    • Epitope Mapping: Prioritize antibodies targeting the C-terminal region (aa 720–848), as this domain is critical for oligomerization and chaperone activity . For N-terminal studies, use antibodies against residues 1–113, though this region is dispensable for ATPase function .

    • Validation: Perform immunoblotting with M. tuberculosis lysates and deletion mutants (e.g., ClpC1–746 or ClpC1–Δ3) to confirm specificity .

    • Table 1: Epitope-specific antibody performance:

      Target Domain	Antibody Clone	Cross-Reactivity (Non-TB Species)	Key Application
      C-terminal	Anti-ClpC1-C	M. smegmatis, M. bovis	Oligomerization assays
      N-terminal	Anti-ClpC1-N	None observed	Localization studies

• What controls are essential for CLPC1 antibody validation in Western blotting?

  • Methodology:

    • Include clpC1 knockdown strains (e.g., M. smegmatis ΔclpC1) as negative controls .

    • Use recombinant ClpC1 (full-length vs. truncated mutants) to confirm antibody binding .

    • Pre-absorption with purified ClpC1 protein to rule out non-specific bands.

Advanced Research Questions

• How do I resolve contradictions in CLPC1 functional data when using domain-specific antibodies?

  • Case Study:

    • Observation: C-terminal deletions (e.g., ClpC1–746) retain ATPase activity but lose chaperone function .

    • Troubleshooting:

      1. Use ATPase activity assays (e.g., malachite green) alongside co-immunoprecipitation (Co-IP) with substrates like Hsp20 .

      2. Pair anti-ClpC1-C antibodies with size-exclusion chromatography to detect oligomerization defects .

    • Key Insight: Antibodies targeting the C-terminus may fail to detect functional defects if oligomerization is disrupted but ATPase domains remain intact .

• What methods optimize CLPC1 detection in intracellular infection models?

  • Methodology:

    • Macrophage Assays: Infect THP-1 cells with M. tuberculosis at MOI 1:2, lyse at 24–72h, and use RIPA buffer with protease inhibitors .

    • Antibody Validation: Compare CLPC1 levels in wild-type vs. clpC1(−) strains via quantitative Western blotting .

    • Table 2: Antibody performance in infection models:

      Application	Antibody	Sensitivity (LOD)	Compatible Assays
      Intracellular Mtb	Anti-ClpC1-C	10 ng/mL	IF, Flow cytometry
      Bacterial lysates	Anti-ClpC1-N	5 ng/mL	Western blot, ELISA

• How do I design experiments to study CLPC1 degradation via BacPROTACs?

  • Protocol:

    1. Treat M. smegmatis or M. tuberculosis with cyclomarin-derived BacPROTACs (e.g., 10 µM for 6h) .

    2. Use anti-ClpC1-C antibodies to monitor degradation via Western blotting (reduced band intensity at ~95 kDa) .

    3. Validate with ATPase activity assays (increased activity precedes degradation) .

Technical Challenges & Solutions

• Why do some CLPC1 antibodies fail in immunoprecipitation (IP) despite working in Western blotting?

  • Root Cause: Conformational sensitivity—antibodies may not bind when ClpC1 is complexed with ClpP1P2 or substrates .

  • Solution:

    • Use crosslinkers (e.g., DSS) before IP to stabilize interactions.

    • Test buffer conditions: 1% Triton X-100 + 2 mM ATP preserves oligomeric states .

• How to distinguish CLPC1 isoforms in M. tuberculosis vs. non-pathogenic mycobacteria?

  • Strategy:

    • Leverage sequence divergence in the C-terminal tail (e.g., M. tuberculosis-specific DVDN motif at aa 802) .

    • Develop custom antibodies against the unconserved 42-aa tail (commercially unavailable; requires peptide immunization) .

Data Interpretation Guidelines

• Critical Controls: Always include clpC1 deletion mutants and ATPase-negative strains to contextualize antibody-derived data .

• Quantitative Analysis: Normalize CLPC1 levels to housekeeping proteins (e.g., GroEL) in infection models to account for bacterial load variations .