clpP1 Antibody

What is ClpP1 and what is its function in bacterial systems?

ClpP1 is a subunit of the caseinolytic peptidase P (ClpP) proteolytic complex, which plays crucial roles in protein homeostasis and regulatory proteolysis. In bacteria like Mycobacterium tuberculosis, the ClpP system consists of discrete ClpP1 and ClpP2 rings that together form a functional proteolytic core . ClpP cleaves peptides in various proteins in a process that requires ATP hydrolysis, typically functioning as a general housekeeping mechanism rather than targeting specific substrates . The ClpP system collaborates with AAA+ (ATPases Associated with diverse cellular Activities) partner proteins such as ClpX and ClpC to carry out energy-dependent protein degradation within cells . In some organisms like Streptomyces, there are multiple clpP genes organized in operons, indicating complex regulation of this proteolytic system .

What are the recommended dilutions for ClpP antibodies in different applications?

Based on validated protocols, the recommended dilutions for ClpP antibodies vary by application and specific antibody clone. For polyclonal antibodies like 15698-1-AP:

For monoclonal antibodies like 66271-1-Ig:

It is strongly recommended that researchers optimize these dilutions for their specific experimental systems to obtain optimal results, as reactivity may be sample-dependent .

How can I validate the specificity of a ClpP1 antibody?

Validating antibody specificity is crucial for reliable research outcomes. For ClpP1 antibodies, a comprehensive validation approach should include:

• Knockdown/Knockout Controls: Using ClpP1 knockdown or knockout samples as negative controls. Several publications have utilized this approach to confirm antibody specificity, with three publications for polyclonal antibody 15698-1-AP and one for monoclonal antibody 66271-1-Ig reporting KD/KO validations .

• Multiple Detection Methods: Confirming consistent results across different techniques. For example, if an antibody detects a protein of the expected molecular weight in Western blot (26 kDa and 30 kDa for ClpP), it should also show appropriate localization patterns in immunofluorescence studies .

• Cross-Species Reactivity: Testing the antibody against samples from different species to confirm conservation of epitope recognition. ClpP antibodies have demonstrated reactivity with human, mouse, rat, and other species, though specific reactivity profiles differ between antibody clones .

• Multiple Antibody Comparison: Using different antibodies targeting distinct epitopes of ClpP1 to confirm consistent detection patterns.

• Peptide Competition Assay: Pre-incubating the antibody with the immunizing peptide should abolish specific signals if the antibody is truly specific.

This multi-faceted approach provides robust validation of ClpP1 antibody specificity for research applications.

What tissue samples have been validated for ClpP antibody applications?

ClpP antibodies have been validated in numerous tissue and cell types across different applications. For Western blot applications, polyclonal antibody 15698-1-AP has been successfully tested in human placenta tissue, rat heart tissue, mouse heart tissue, and K-562 cells . The monoclonal antibody 66271-1-Ig has shown positive Western blot results in human heart tissue and pig heart tissue .

For immunohistochemistry, positive detection has been confirmed in human liver cancer tissue, human kidney tissue, human testis tissue, and human liver tissue using polyclonal antibodies . The monoclonal antibody has been validated for IHC in human heart tissue .

Immunoprecipitation applications have been validated using mouse skeletal muscle tissue, while immunofluorescence applications have been confirmed in HeLa cells . This diverse validation across tissues and cell types provides researchers with confidence when selecting appropriate samples for their ClpP studies.

How does the heteromeric ClpP1P2 complex in Mycobacterium tuberculosis function, and why is it a potential drug target?

The ClpP1P2 complex in Mycobacterium tuberculosis represents a unique heteromeric structure essential for the pathogen's survival. Unlike ClpP systems in many other bacteria that form homomeric complexes, M. tuberculosis requires both ClpP1 and ClpP2 rings to create a functional proteolytic core .

Crystal structure analysis at 3.20 Å resolution (PDB ID code 4U0G) revealed that the ClpP1P2 complex consists of discrete ClpP1 and ClpP2 rings that associate to form a functional proteolytic barrel . The heteromeric nature of this complex creates distinct binding sites and catalytic mechanisms that differ from homologous systems in other bacteria.

The ClpP1P2 system is particularly important as a drug target because:

• It is essential for the survival of M. tuberculosis, making it a validated target for therapeutic intervention.

• The unique heteromeric structure provides opportunities for selective targeting that may not affect human proteases.

• Small molecules like acyldepsipeptides (ADEPs) can bind to ClpP and cause dysregulation of the proteolytic function, leading to uncontrolled proteolysis and bacterial death.

Understanding the structural basis of the ClpP1P2 complex provides a foundation for rational drug design targeting this essential protease system in M. tuberculosis .

What is the mechanism of acyldepsipeptide (ADEP) interaction with the ClpP system?

Acyldepsipeptides (ADEPs) represent an important class of antibiotics that target the ClpP proteolytic system. Their mechanism of action is multi-layered and involves:

• Binding Specificity: In Streptomyces, ADEP selectively binds to ClpP1 but not ClpP2, creating a targeted dysregulation of the proteolytic system .

• Unregulated Proteolysis: When ADEP binds to ClpP1, it triggers the degradation of non-native protein substrates in an uncontrolled manner, bypassing the normal regulatory mechanisms that require AAA+ partners .

• Dual Binding Mechanism: Remarkably, ADEP and Clp-ATPases can simultaneously bind to opposite sides of the ClpP1P2 barrel. This concomitant binding results in a third mechanism of ADEP action: the accelerated proteolysis of native protein substrates by the Clp protease complex .

• Structural Basis: Crystal structure analysis of ClpP1P2 bound to ADEP (PDB ID code 4U0G) has provided insights into the molecular interactions that facilitate this dysregulation .

This multilayered mechanism explains why ADEP compounds show promise for treating multiresistant and persistent bacterial infections. The ability to cause both unregulated proteolysis of non-native proteins and accelerated degradation of natural Clp protease substrates makes ADEP a powerful disruptor of bacterial proteostasis .

How can I optimize Co-Immunoprecipitation protocols using ClpP1 antibodies to study protein interactions?

Optimizing Co-Immunoprecipitation (Co-IP) protocols with ClpP1 antibodies requires careful consideration of several factors to preserve native protein-protein interactions while achieving specific pulldown. Based on published applications and technical data, the following optimization strategies are recommended:

• Antibody Selection: Choose an antibody validated for IP applications. The polyclonal antibody 15698-1-AP has been documented in successful IP applications, with a recommended usage of 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate .

• Lysis Buffer Optimization: Use gentle lysis conditions that preserve protein-protein interactions. A buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, and protease inhibitors is often suitable for ClpP complex studies. Avoid harsh detergents like SDS that disrupt protein interactions.

• Cross-linking Considerations: For transient or weak interactions, consider using a reversible cross-linking agent like DSP (dithiobis[succinimidyl propionate]) before cell lysis to stabilize protein complexes.

• Preclearing Step: To reduce non-specific binding, preclear lysates with protein A/G beads before adding the ClpP1 antibody.

• Incubation Conditions: Perform antibody binding at 4°C overnight with gentle rotation to maximize specific interactions while minimizing non-specific binding.

• Washing Stringency: Balance between removing non-specific interactions and preserving genuine interactions by optimizing salt concentration and detergent levels in wash buffers.

• Elution Strategy: Consider native elution using excess immunizing peptide if the antibody epitope is known, or use a gentle acidic elution to preserve co-precipitated proteins.

• Controls: Include appropriate negative controls (non-specific IgG, lysate from ClpP1 knockout cells) and positive controls (known interacting partners like ClpX or ClpC) to validate results .

By systematically optimizing these parameters, researchers can develop robust Co-IP protocols for studying ClpP1 interactions with partner proteins like ClpX, ClpC, or other components of the protein quality control machinery.

How can ClpP1 antibodies be used to investigate the role of ClpP in bacterial antibiotic resistance?

ClpP1 antibodies serve as valuable tools for investigating the role of ClpP in bacterial antibiotic resistance through several methodological approaches:

• Expression Level Analysis: Western blot analysis using ClpP1 antibodies can quantify changes in ClpP expression levels in response to antibiotic treatment or in resistant strains compared to susceptible ones. The recommended dilutions of 1:1000-1:8000 for polyclonal antibodies or 1:500-1:2000 for monoclonal antibodies provide a strong signal-to-noise ratio for quantitative analysis .

• Localization Studies: Immunofluorescence microscopy using ClpP1 antibodies at dilutions of 1:50-1:500 can reveal changes in the subcellular localization of ClpP in response to antibiotics, potentially identifying novel mechanisms of resistance .

• Protein-Protein Interaction Networks: Co-immunoprecipitation using ClpP1 antibodies followed by mass spectrometry can identify changes in the interactome of ClpP in resistant versus susceptible bacteria, potentially revealing new resistance mechanisms involving altered protein quality control pathways.

• Post-translational Modifications: Immunoprecipitation of ClpP1 followed by analysis with modification-specific antibodies can determine whether post-translational modifications of ClpP contribute to resistance phenotypes.

• ClpP Activity Modulation Studies: ClpP1 antibodies can be used to monitor how compounds like acyldepsipeptides (ADEPs) interact with and modulate ClpP activity. Since ADEPs bind to ClpP1 (but not ClpP2) in Streptomyces and dysregulate proteolysis, antibodies can help track these interactions and their consequences for antibiotic sensitivity .

• Structural Analysis Support: While crystal structures provide high-resolution information about ClpP1P2 complexes , antibodies can complement these studies by confirming the presence and composition of these complexes in living cells under various antibiotic stress conditions.

By implementing these approaches, researchers can gain deeper insights into how modulation of the ClpP system contributes to antibiotic resistance and potentially identify novel strategies to combat resistant infections.

What approaches are recommended for studying ClpP1-ClpC1 interactions in mycobacteria?

Studying ClpP1-ClpC1 interactions in mycobacteria requires specialized approaches due to the unique heteromeric ClpP1P2 complex and the specific nature of AAA+ partner interactions. Based on current research methodologies, the following approaches are recommended:

• Recombinant Protein Expression Systems: Establishing E. coli-based overexpression systems for both ClpP1 and ClpC1 has proven successful for interaction studies. Western blot analysis of purified ClpP1 has shown multiple bands (60kDa, 40kDa, and the expected size), possibly representing uncleaved and cleaved forms, which should be considered when interpreting results .

• ATPase Activity Assays: ClpC1 displays basal ATPase activity without requiring other chaperones or adaptor proteins. This activity can be measured and used as a functional readout of ClpC1 interaction with ClpP1 or with small molecule modulators like Compound X, which has been shown to enhance ClpC1 ATPase activity .

• Structural Studies: The crystal structure of ClpP1P2 from M. tuberculosis at 3.20 Å resolution (PDB ID code 4U0G) provides a framework for understanding the structural basis of interactions. Complementary approaches like cryo-EM could reveal the dynamic aspects of ClpP1-ClpC1 interactions .

• Co-Immunoprecipitation: Using ClpP1 antibodies for Co-IP followed by detection of ClpC1 can confirm interactions in native or near-native conditions. The recommended antibody amount is 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate .

• Proteolytic Activity Assays: Since ClpP1 requires ClpP2 to form a functional proteolytic core in mycobacteria, assays measuring the degradation of model substrates in the presence of ClpC1 can provide functional evidence of productive interactions .

• Small Molecule Modulation: Compounds like Compound X and its derivatives that specifically enhance ClpC1 ATPase activity can be used as chemical probes to understand the functional consequences of ClpP1-ClpC1 interactions .

• In Vivo Crosslinking: Chemical crosslinking followed by immunoprecipitation with ClpP1 antibodies can capture transient interactions between ClpP1 and ClpC1 in their native cellular environment.

These complementary approaches provide a comprehensive toolkit for investigating the molecular basis and functional significance of ClpP1-ClpC1 interactions in mycobacteria, potentially leading to new therapeutic strategies targeting this essential system.

What are the optimal conditions for antigen retrieval when using ClpP1 antibodies in immunohistochemistry?

Successful immunohistochemical detection of ClpP1 requires careful optimization of antigen retrieval conditions. Based on validated protocols, the following recommendations should be considered:

For both polyclonal (15698-1-AP) and monoclonal (66271-1-Ig) ClpP antibodies, the primary recommended antigen retrieval method is:

• Primary Recommendation: TE buffer at pH 9.0

  • This alkaline pH helps disrupt protein cross-links formed during fixation, particularly for formalin-fixed, paraffin-embedded (FFPE) tissues

  • The TE buffer typically consists of 10 mM Tris Base, 1 mM EDTA, and 0.05% Tween 20, adjusted to pH 9.0

• Alternative Method: Citrate buffer at pH 6.0

  • This can be used as an alternative when TE buffer retrieval is suboptimal

  • Typically prepared as 10 mM sodium citrate buffer with 0.05% Tween 20, adjusted to pH 6.0

• Retrieval Process:

  • Heat-induced epitope retrieval (HIER) methods are recommended

  • Tissue sections should be immersed in the retrieval buffer

  • Heating can be performed using a pressure cooker, microwave, or water bath

  • For pressure cooker: 3-5 minutes at full pressure

  • For microwave: 10-20 minutes at medium power

  • For water bath: 20-40 minutes at 95-98°C

These conditions have been validated with various human tissues including liver cancer tissue, kidney tissue, testis tissue, liver tissue, and heart tissue . The choice between TE buffer and citrate buffer may depend on the specific tissue being examined and should be empirically determined for each new tissue type or fixation condition.

How should researchers address discrepancies in molecular weight observations for ClpP in Western blot analyses?

When working with ClpP antibodies in Western blot applications, researchers often encounter molecular weight discrepancies that require careful interpretation:

• Expected vs. Observed Weights: Both polyclonal and monoclonal ClpP antibodies report a calculated molecular weight of 30 kDa, but observed molecular weights are typically 26 kDa and 30 kDa . This dual banding pattern is consistent across different antibody clones and sample types.

• Causes of Discrepancies:

  • Protein Processing: The presence of both 26 kDa and 30 kDa bands likely represents different processing states of ClpP. The protein undergoes cleavage of a mitochondrial targeting sequence upon import into mitochondria.

  • Post-translational Modifications: Various modifications can alter migration patterns.

  • Isoforms: Multiple ClpP isoforms may be detected depending on the epitope recognized by the antibody.

• Methodological Solutions:

  • Positive Controls: Include samples with known ClpP expression patterns, such as human placenta tissue, rat heart tissue, or mouse heart tissue .

  • Knockdown/Knockout Validation: Use ClpP knockdown or knockout samples to confirm band specificity.

  • Optimized Sample Preparation: Complete denaturation and reduction of samples is critical. Use fresh sample buffer with reducing agents and heat samples at 95°C for 5 minutes.

  • Gradient Gels: Consider using gradient gels (4-15% or 4-20%) for better resolution of closely migrating bands.

  • Extended Separation Time: Longer electrophoresis times can improve separation of the 26 kDa and 30 kDa bands.

  • Multiple Antibody Comparison: Using both monoclonal and polyclonal antibodies targeting different epitopes can help validate which bands represent authentic ClpP.

• Interpretation Guidelines:

  • In mycobacteria studies, purified ClpP1 has shown additional bands at 60 kDa and 40 kDa, possibly representing uncleaved and partially cleaved forms . Researchers should be aware of these potential higher molecular weight forms.

  • When quantifying ClpP levels, consider including both the 26 kDa and 30 kDa bands in densitometric analyses unless there is clear evidence that only one represents the protein of interest.

By applying these methodological approaches, researchers can address molecular weight discrepancies and ensure accurate interpretation of Western blot results when studying ClpP.