CLPB1 Antibody

What is CLPB and what are its primary cellular functions?

CLPB (also known as SKD3 or Mitochondrial disaggregase) functions as a regulatory ATPase with ATP-dependent protein disaggregase activity. This protein plays several critical roles in cellular function, including maintaining the solubility of key mitochondrial proteins and participating in secretion/protein trafficking processes. CLPB is involved in mitochondrial-mediated antiviral innate immunity, where it activates RIG-I-mediated signal transduction and production of IFNB1 and pro-inflammatory cytokine IL6. Additionally, it plays a role in granulocyte differentiation .

CLPB is a member of the AAA+ protein family, encoding a protein of 707 amino acids with a predicted molecular mass of approximately 79 kDa. It contains two conserved ATP-binding domains that are essential for its disaggregase function . The protein's disaggregase activity is particularly important for maintaining the functional integrity of the mitochondrial intermembrane space proteome.

What detection methods are compatible with CLPB antibodies?

CLPB antibodies have been validated for multiple research applications, with varying degrees of optimization required:

For optimal results, researchers should titrate antibodies in their specific experimental systems, as sensitivity can vary based on sample type and detection method . When using commercially available antibodies, validation in your experimental system is crucial for reliable results.

What differences should researchers expect between predicted and observed molecular weights of CLPB?

While CLPB has a calculated molecular weight of 79 kDa (707 amino acids), researchers frequently observe bands at approximately 58 kDa in Western blot analyses . This discrepancy may be attributed to several factors:

• Potential internal translation initiation sites that could produce a shorter form of the protein

• Post-translational modifications affecting protein migration

• Proteolytic processing in specific cellular compartments

When analyzing Western blot results, researchers should be prepared for this discrepancy and include appropriate positive controls. If multiple bands are observed, validation with knockout controls or other techniques may be necessary to confirm specificity .

What are the optimal conditions for using CLPB antibodies in Western blotting?

For effective Western blot detection of CLPB, researchers should consider the following protocol:

• Sample preparation: Use cell pellets from sufficient culture volume (e.g., 200 ml) to ensure adequate protein yield.

• Buffer selection: 100 mM phosphate-buffered saline (PBS; 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.0) has been used successfully.

• Blocking: Fat-free milk serves as an effective blocking agent.

• Antibody dilutions: Primary antibody at 1:5,000 dilution and alkaline phosphatase-conjugated secondary antibody at 1:3,000 dilution.

• Development: Use an alkaline phosphatase substrate kit for visualization.

• Quantification: Image analysis software like ImageJ can be used for band intensity determination .

When comparing CLPB expression levels between samples, loading controls should be carefully selected, especially when working with mitochondrial proteins where traditional housekeeping proteins may not be appropriate.

How should immunohistochemistry protocols be optimized for CLPB detection?

For successful immunohistochemical analysis of CLPB:

• Antigen retrieval: Use TE buffer at pH 9.0, although citrate buffer at pH 6.0 may also be effective for certain tissues.

• Antibody concentration: Start with a 1:100 dilution for paraffin-embedded tissues, adjusting as needed.

• Tissue selection: CLPB antibodies have been successfully used on human adrenal gland and heart tissues.

• Visualization: Standard DAB (3,3'-diaminobenzidine) or other chromogenic substrates are suitable .

When examining CLPB localization, pay particular attention to mitochondria-rich regions of the tissue, as CLPB function is closely linked to mitochondrial proteostasis. Positive and negative controls should be included to ensure specificity of staining.

What approaches can be used to validate CLPB antibody specificity?

To ensure the specificity of CLPB antibodies, researchers should implement the following validation strategies:

• CRISPR-Cas9 knockout controls: Generate CLPB knockout cell lines using CRISPR-Cas9 genome editing to serve as negative controls .

• siRNA knockdown: As an alternative to complete knockout, siRNA-mediated knockdown can reduce CLPB expression.

• Overexpression systems: Complementary to knockout approaches, overexpression of tagged CLPB can confirm antibody specificity.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to block specific binding.

• Cross-validation: Use multiple antibodies targeting different epitopes of CLPB.

Each validation approach offers advantages and limitations, and researchers should select methods based on their experimental context and available resources.

How can CLPB antibodies be used to investigate mitochondrial protein disaggregation mechanisms?

To study CLPB's role in mitochondrial protein disaggregation:

• Subcellular fractionation: Isolate mitochondria to examine CLPB's association with this organelle under various stress conditions.

• Co-immunoprecipitation: Use CLPB antibodies to identify protein interaction partners, particularly under stress conditions that promote protein aggregation.

• Immunofluorescence co-localization: Combine CLPB staining with markers for protein aggregates to visualize disaggregation activities.

• Stress induction experiments: Apply heat shock or oxidative stress to cells and monitor CLPB distribution and protein aggregate formation.

Research has shown that CLPB dysfunction leads to aggregation of quality control proteins in the intermembrane space. When designing experiments to study this phenomenon, consider using label-free quantitative mass spectrometry on isolated mitochondria from wild-type and CLPB knockout cells to identify proteins affected by CLPB deficiency .

What methods can be used to study CLPB's role in heat shock response?

Building on findings that ClpB1 overproduction increases temperature tolerance in Synechocystis:

• Generate overexpression systems: Create stable cell lines overexpressing wild-type CLPB and ATPase-deficient mutants.

• Heat shock protocols: Apply gradual or rapid temperature increases (e.g., 1°C/s to 50°C) to study differential effects.

• Cell survival assays: Measure cell viability after heat shock using standard viability assays.

• Protein aggregation analysis: Use electron microscopy to visualize and quantify protein aggregates in control versus CLPB-overexpressing cells.

• Co-expression studies: Investigate synergistic effects by co-overexpressing CLPB with other heat shock proteins like DnaK2 .

When designing heat shock experiments, consider both the rate of temperature increase and the final temperature, as these parameters can significantly affect cellular responses and the protective capacity of CLPB.

How can CLPB antibodies help investigate mitochondrial quality control mechanisms?

To examine CLPB's role in mitochondrial quality control:

• Monitor OPA1 processing: Western blot analysis can reveal changes in OPA1 processing patterns, which are altered in CLPB-deficient cells.

• Assess mitochondrial morphology: Use immunofluorescence to visualize changes in mitochondrial network architecture.

• Measure reactive oxygen species (ROS): Quantify ROS production as an indicator of mitochondrial dysfunction.

• Analyze OXPHOS components: Examine the abundance and assembly of respiratory chain complexes in the presence and absence of CLPB.

Research has demonstrated that CLPB knockout leads to excessive OPA1 processing and accumulation of short-form OPA1, which can be reversed by re-expression of wild-type CLPB but not ATPase-deficient mutants . This suggests CLPB plays a critical role in maintaining mitochondrial inner membrane dynamics.

What approaches can identify the role of CLPB in antiviral immunity mechanisms?

To investigate CLPB's involvement in mitochondrial-mediated antiviral innate immunity:

• Viral infection models: Challenge cells with RNA viruses and monitor CLPB expression and localization.

• RIG-I pathway activation: Measure the activation of RIG-I and downstream signaling molecules in the presence and absence of CLPB.

• Cytokine production: Quantify IFNB1 and IL6 production following viral challenge in control versus CLPB-deficient cells.

• Protein interaction studies: Identify interactions between CLPB and components of the antiviral signaling machinery.

When designing these experiments, consider the timing of sample collection, as antiviral responses occur in distinct phases. Additionally, using multiple viral stimuli can help distinguish CLPB's specific role in different antiviral pathways .

How can researchers troubleshoot non-specific binding in CLPB antibody applications?

When encountering non-specific binding issues:

• Optimize blocking conditions: Test different blocking agents (BSA, casein, normal serum) and concentrations.

• Adjust antibody concentration: Titrate primary antibody dilutions to find optimal signal-to-noise ratio.

• Modify washing protocols: Increase washing stringency by adding detergents or salt to buffers.

• Pre-adsorb antibodies: Incubate with non-target tissue lysates to remove cross-reactive antibodies.

• Use knockout controls: Include CLPB knockout samples to identify non-specific bands.

For especially problematic samples, consider using monoclonal antibodies instead of polyclonal ones, as they typically offer higher specificity at the cost of potentially lower sensitivity.

What controls are essential when studying CLPB localization using immunofluorescence?

For reliable immunofluorescence localization studies:

• Include mitochondrial markers: Co-stain with established mitochondrial markers to confirm organelle localization.

• Use knockout controls: Include CLPB-deficient cells to establish staining specificity.

• Add peptide competition controls: Block antibody binding with immunizing peptide.

• Include secondary-only controls: Evaluate background from secondary antibodies.

• Test multiple fixation methods: Different fixatives may preserve CLPB epitopes differently.

Research suggests CLPB may concentrate in specific areas near membranes, similar to observations in Synechocystis where ClpB1 was dispersed throughout the cytoplasm but concentrated near thylakoid membranes . This nuanced distribution pattern requires careful imaging and quantification.

How should researchers interpret CLPB expression changes in disease models or stress conditions?

When analyzing CLPB expression changes:

• Consider post-translational modifications: Changes in function may occur without changes in total protein levels.

• Assess subcellular distribution: Relocalization may indicate functional changes independent of expression levels.

• Evaluate protein interactions: Changes in binding partners may indicate altered function.

• Measure disaggregase activity: Functional assays can complement expression data.

• Correlate with phenotypic outcomes: Connect expression changes to cellular responses and phenotypes.

Proteomic approaches can provide comprehensive insights into how CLPB dysfunction affects the mitochondrial proteome. Label-free quantitative mass spectrometry has revealed that CLPB knockout leads to increased abundance of pro-apoptotic factors and depletion of respiratory chain components .

What methodologies can distinguish between CLPB's direct and indirect effects on mitochondrial function?

To differentiate direct from indirect effects:

• Structure-function studies: Generate point mutations in specific CLPB domains to dissect their contributions.

• Temporal analysis: Use inducible expression or degradation systems to observe immediate versus delayed effects.

• Substrate identification: Employ techniques like BioID or APEX proximity labeling to identify direct CLPB interactors.

• In vitro disaggregation assays: Reconstitute CLPB activity with purified components to verify direct effects.

Research has shown that CLPB dysfunction leads to excessive ROS accumulation and damage to electron transport chain components, suggesting both direct effects on protein disaggregation and indirect consequences for mitochondrial function .

How can integrated multi-omics approaches enhance our understanding of CLPB function?

For comprehensive understanding of CLPB biology:

• Combine proteomics, transcriptomics, and metabolomics: Integrate data from multiple -omics platforms.

• Perform temporal profiling: Track changes across multiple time points after CLPB manipulation.

• Analyze PTMs: Examine post-translational modifications that may regulate CLPB function.

• Conduct interactome studies: Map the network of CLPB interactions under different conditions.

This integrated approach can reveal how CLPB dysfunction cascades through cellular systems, affecting processes beyond immediate disaggregation targets. For example, studies have shown connections between CLPB and OPA1 processing, suggesting broader impacts on mitochondrial dynamics .