CLPX2 Antibody

What is CLPP and what function does it serve in cellular processes?

CLPP (Endopeptidase Clp) is a protease component of the Clp complex that cleaves peptides and various proteins in an ATP-dependent process. It functions as a critical part of the mitochondrial protein quality control system. CLPP exhibits low peptidase activity independently but significantly higher activity when associated with CLPX, forming the complete Clp complex. This complex is responsible for degrading multiple substrates including CSN1S1, CSN2, and CSN3, as well as synthetic peptides in vitro . Rather than targeting specific substrates exclusively, CLPP appears to serve a central housekeeping function in cellular protein maintenance. One notable specific substrate is PINK1, which CLPP cleaves within the mitochondrion, suggesting its involvement in pathways related to Parkinson's disease .

How does ClpC2 differ from ClpC1 in mycobacterial species?

ClpC2 is a partial homologue of ClpC1 that plays a crucial role in mycobacterial antibiotic resistance. While both proteins belong to the same family, they serve distinct functions. ClpC1 is an essential component of the mycobacterial Clp protease system involved in protein degradation, whereas ClpC2 functions as a protective mechanism against certain antibiotics, particularly Cyclomarin A (CymA) .

Unlike ClpC1, whose expression remains relatively constant regardless of antibiotic exposure, ClpC2 shows dramatic upregulation (up to 263-fold increase in mRNA levels) following exposure to CymA . Functionally, ClpC2 acts as a molecular sponge by binding to CymA, thus preventing the antibiotic from targeting the essential ClpC1 protein. This protective role allows mycobacteria to evade the toxic effects of CymA by sequestering the antibiotic away from its primary target .

What are the optimal protocols for validating CLPP antibody specificity?

Validating CLPP antibody specificity requires a multi-faceted approach:

• Knockout/Knockdown Validation: Generate CLPP-deficient samples using CRISPR-Cas9 or siRNA techniques. A specific antibody should show significantly reduced or absent signal in these samples compared to wild-type controls. Published literature contains at least 3 studies utilizing CLPP knockdown/knockout validation .

• Cross-reactivity Testing: Test the antibody against related Clp family proteins to ensure specificity. This is particularly important as Clp proteins share structural similarities.

• Multiple Application Validation: Confirm specificity across different applications (WB, IHC, IF/ICC):

  • For Western blot: Use 1:1000-1:8000 dilution and verify the 26-30 kDa bands

  • For IHC: Use 1:50-1:500 dilution with TE buffer (pH 9.0) for antigen retrieval

  • For IF/ICC: Use 1:50-1:500 dilution and compare subcellular localization to known mitochondrial patterns

• Multiple Species Testing: CLPP antibodies have demonstrated reactivity with human, mouse, and rat samples, allowing for comparative studies across species .

These validation approaches collectively provide robust evidence of antibody specificity, essential for generating reliable experimental data.

How does ClpC2 expression change in response to antibiotic exposure, and what techniques best capture this dynamic?

ClpC2 exhibits remarkable expression plasticity in response to Cyclomarin A (CymA). Quantitative analysis reveals:

• Transcript Level Changes: RT-qPCR analysis shows that ClpC2 mRNA increases 263-fold after just one hour of exposure to sub-MIC50 concentrations of CymA .

• Protein Level Dynamics: Western blot analysis demonstrates that ClpC2 protein, initially undetectable in untreated mycobacteria, increases dramatically over 8 hours following CymA exposure. Quantitative Western blotting reveals ClpC2 reaches levels 84-fold higher than ClpC1 protein levels by the 8-hour timepoint .

• Recommended Technical Approach:

  • For capturing the full dynamic range of expression: Use a time-course experiment combining RT-qPCR and quantitative Western blotting

  • For protein:protein comparisons: Use standardized loading controls (e.g., RpoB) and recombinant protein standards for absolute quantification

  • For mechanism studies: Complement with DNA-protein binding assays (as ClpC2 appears to regulate its own promoter)

This dramatic upregulation represents a sophisticated bacterial defense mechanism where ClpC2 acts as a molecular sponge, sequestering the antibiotic away from essential ClpC1 protease components.

What experimental approaches can determine if CLPP is involved in a specific proteolytic pathway?

Determining CLPP involvement in specific proteolytic pathways requires a multi-method approach:

• Substrate Identification:

  • Co-immunoprecipitation using CLPP antibodies (0.5-4.0 μg antibody per 1.0-3.0 mg total protein lysate)

  • Mass spectrometry analysis of CLPP-associated proteins

  • In vitro degradation assays with recombinant CLPP and candidate substrates

• Degradation Kinetics Analysis:

  • Pulse-chase experiments with potential substrates in the presence/absence of CLPP

  • FITC-casein degradation assays, which have been successfully used to study Clp protease activity

  • Time-course analysis of substrate levels following CLPP manipulation

• Genetic Manipulation Approaches:

  • CLPP knockout/knockdown followed by proteome-wide analysis

  • Complementation studies using wild-type versus mutant CLPP

  • Analysis of substrate accumulation in CLPP-deficient models

• Pharmacological Intervention:

  • Use of specific Clp protease inhibitors

  • Monitoring substrate levels following inhibitor treatment

  • Comparison with genetic manipulation results to confirm specificity

The combined results from these approaches provide strong evidence for CLPP involvement in specific proteolytic pathways, as exemplified by the confirmation of PINK1 as a CLPP substrate in mitochondria .

What are the recommended antibody dilutions and conditions for different CLPP detection applications?

Optimal detection of CLPP across different experimental applications requires specific antibody dilutions and conditions:

Important considerations for optimal results:

• For IHC applications, while TE buffer (pH 9.0) is recommended for antigen retrieval, citrate buffer (pH 6.0) may be used as an alternative

• Antibody dilution should be optimized for each experimental system and sample type

• For Western blot, researchers should expect bands at both 26 kDa and 30 kDa

• For co-immunoprecipitation studies targeting CLPP-associated proteins, published literature provides validated protocols

How can the fusion protein approach enhance antibody generation for Clp protein complexes?

The fusion protein approach represents an innovative method for generating antibodies against protein complexes that are otherwise difficult to target:

• Methodological Approach:

  • Design a fusion construct combining the interacting domains of Clp complex proteins

  • Express and purify the stabilized fusion protein

  • Use the fusion protein as an immunogen for antibody production

  • Screen resulting antibodies for specificity to the protein complex interface

• Advantages for Clp Complex Research:

  • Overcomes the instability issues common in protein complexes during immunization

  • Enables generation of antibodies specific to the protein-protein interface

  • Allows direct measurement of complex formation on live cells

  • Facilitates distinguishing between free individual proteins and their complexed forms

• Implementation for Clp Proteins:

  • This approach could be particularly valuable for generating antibodies against CLPP-CLPX complexes

  • The method has been successfully demonstrated with the BTLA-HVEM complex, showing potential transferability to other protein complexes

  • The resulting antibodies enable quantification of the ratio between free proteins and their complexed forms in cellular systems

This approach addresses a critical gap in current antibody technology by enabling specific targeting of functional protein complexes rather than individual proteins alone, opening new research avenues for studying Clp protease function in various cellular contexts.

What experimental designs can effectively measure the functional impact of CLPP in mitochondrial proteostasis?

Effective experimental designs to measure CLPP's functional impact on mitochondrial proteostasis include:

• Mitochondrial Substrate Accumulation Assays:

  • Compare levels of known CLPP substrates (e.g., PINK1) in CLPP-sufficient versus CLPP-deficient cells

  • Use Western blotting with validated CLPP antibodies (1:1000-1:8000 dilution) to confirm CLPP knockdown/knockout

  • Quantify changes in substrate levels using densitometry or fluorescence-based detection methods

• Organelle-Specific Proteomics:

  • Isolate intact mitochondria from control and CLPP-manipulated cells

  • Perform comparative mass spectrometry to identify accumulated proteins

  • Validate candidates using targeted Western blotting with appropriate controls

• Functional Mitochondrial Assays:

  • Measure oxygen consumption rate (OCR) using Seahorse technology

  • Assess mitochondrial membrane potential using potentiometric dyes

  • Quantify ATP production in the presence/absence of CLPP activity

• Live-Cell Imaging:

  • Use fluorescently-tagged CLPP substrates to monitor degradation kinetics

  • Apply immunofluorescence with CLPP antibodies (1:50-1:500 dilution) to assess localization

  • Implement time-lapse microscopy to capture dynamic changes in substrate levels

• Genetic Complementation Studies:

  • Rescue CLPP-deficient phenotypes with wild-type versus mutant CLPP

  • Use site-directed mutagenesis to disrupt the active site (for mechanistic studies)

  • Analyze restoration of normal mitochondrial proteostasis using the above assays

These experimental approaches collectively provide a comprehensive assessment of CLPP's role in maintaining mitochondrial protein quality control, essential for understanding mitochondrial dysfunction in various disease states.

How might CLPP antibodies be utilized to investigate mitochondrial dysfunction in neurodegenerative diseases?

CLPP antibodies offer valuable tools for investigating mitochondrial dysfunction in neurodegenerative conditions:

• PINK1-Parkinson's Disease Connection:

  • CLPP has been shown to cleave PINK1 in mitochondria, a protein implicated in Parkinson's disease

  • CLPP antibodies can be used to assess CLPP expression and localization in patient-derived samples

  • Co-immunoprecipitation using CLPP antibodies can identify disease-specific interacting partners

• Research Methodology:

  • Post-mortem tissue analysis using immunohistochemistry (1:50-1:500 dilution) to compare CLPP levels and distribution between healthy and diseased brain tissues

  • Patient-derived cell models (fibroblasts, iPSC-derived neurons) analyzed by immunofluorescence (1:50-1:500) and Western blotting (1:1000-1:8000)

  • Proteomics following CLPP immunoprecipitation (0.5-4.0 μg antibody per sample) to identify disease-relevant substrates

• Experimental Design Considerations:

  • Include age-matched controls when analyzing human samples

  • Validate CLPP antibody specificity in neural tissues specifically

  • Combine with functional assays of mitochondrial health (respiration, membrane potential)

  • Consider both acute and chronic models of neurodegeneration

This research direction could reveal novel therapeutic targets by identifying specific substrates that accumulate when CLPP function is compromised in neurodegenerative conditions.

What approaches can determine whether ClpC2 upregulation represents a viable target for overcoming antibiotic resistance?

Investigating ClpC2 as a potential target for antibiotic resistance intervention requires systematic approaches:

• Genetic Manipulation Studies:

  • Create clpC2 knockout strains in clinically relevant mycobacterial species

  • Compare antibiotic susceptibility between wild-type and knockout strains

  • Complement knockouts with various clpC2 mutants to identify critical domains

• Structural Biology Approaches:

  • Determine binding interfaces between ClpC2 and antibiotics using crystallography

  • Design small molecules that could prevent ClpC2-antibiotic interactions

  • Test these molecules in combination with existing antibiotics

• Transcriptional Regulation Analysis:

  • Investigate the mechanism of clpC2 upregulation (263-fold increase) in response to antibiotics

  • Identify regulatory elements controlling the massive autoupregulation

  • Target these regulatory mechanisms to prevent ClpC2 upregulation

• Quantitative Assessment:

  • Measure the stoichiometry between ClpC2 and antibiotics in resistant strains

  • Determine the threshold ratio (84-fold excess of ClpC2 over ClpC1) needed for protection

  • Design intervention strategies to prevent reaching this threshold

These approaches could lead to adjuvant therapies that specifically inhibit ClpC2 upregulation or function, potentially restoring antibiotic sensitivity in resistant mycobacterial strains.

How can newly developed antibody generation methods for protein complexes be applied to study CLPP interactions?

The innovative fusion protein approach for antibody generation can significantly advance research on CLPP interactions:

• Adaptation for CLPP Complex Research:

  • Design fusion proteins combining CLPP with its key interacting partners (e.g., CLPX)

  • Generate antibodies specific to the CLPP-CLPX interface

  • Use these antibodies to quantify the proportion of CLPP engaged in active complexes versus free CLPP

• Technical Implementation:

  • Create a series of fusion constructs with varying linker lengths to optimize epitope presentation

  • Screen resulting antibodies for specificity to the complex versus individual proteins

  • Validate using genetic models with mutations at the protein interaction interface

• Research Applications:

  • Direct measurement of CLPP-containing complexes in living cells under various stress conditions

  • Comparison of complex formation between healthy and diseased tissues

  • Screening for compounds that modulate CLPP complex formation

• Advantages Over Traditional Methods:

  • Enables detection of functional complexes rather than just protein levels

  • Provides spatial information about where complex formation occurs within cells

  • Allows quantification of the dynamic equilibrium between free and complexed forms

This approach represents a significant methodological advancement that could reveal previously inaccessible insights into how CLPP complex formation is regulated in different cellular contexts and disease states.