CPN10-2 Antibody

What is CPN10 and why is it important in research?

CPN10 functions as a co-chaperonin to CPN60 in the mitochondrial matrix, playing a crucial role in proper protein folding. In organisms like Leishmania donovani, CPN10 demonstrates stage-specific expression with significantly increased synthesis during differentiation to the amastigote stage . This differential expression makes it an important target for understanding stress responses and developmental transitions in various cellular systems. CPN10 forms functional complexes with CPN60.2, confirming its role as a bona fide co-chaperonin in eukaryotic mitochondria .

How are CPN10 antibodies typically produced for research applications?

Production of CPN10 antibodies typically involves subcloning the CPN10 open reading frame into expression vectors such as pJC45. The recombinant protein is then expressed in E. coli strains like BL21(DE3), purified via metal chelate chromatography, and used for immunization . For polyclonal antibody production, laying hens have been successfully used as hosts, with antibodies subsequently prepared from egg yolk . Alternative approaches include creating synthetic peptides spanning specific regions (such as residues 1-76) of the CPN10 protein for targeted antibody production .

What subcellular localization should researchers expect when using CPN10 antibodies?

When using CPN10 antibodies for localization studies, researchers should expect primarily mitochondrial signals. In Leishmania studies, immune electron microscopy has definitively demonstrated that CPN10 localizes to both tubular mitochondrial structures and the kinetoplast . Fluorescence microscopy using CPN10::GFP chimeras confirms this localization pattern, showing bright fluorescence in tubular structures with no cytoplasmic signal . This mitochondrial localization is consistent with CPN10's role as a co-chaperonin in the mitochondrial matrix.

How can CPN10 antibodies be optimized for co-immunoprecipitation studies?

For successful co-immunoprecipitation of CPN10 with its binding partners, researchers should consider the following methodological approach: prepare cell lysates under non-denaturing conditions, incubate with anti-CPN10 antibodies followed by secondary antibodies (such as anti-chicken IgG), and precipitate complexes using Protein-A agarose . This approach has been successfully used to demonstrate the interaction between CPN10 and CPN60.2 in Leishmania donovani . When analyzing precipitates, immunoblotting with antibodies against suspected binding partners (such as anti-CPN60.2) will confirm interactions. Optimization may include adjusting buffer conditions, antibody concentrations, and incubation times to maximize specific interactions while minimizing background.

What are the best methods for quantifying stage-specific expression of CPN10 using antibodies?

For accurate quantification of stage-specific CPN10 expression, immunoblot analysis of cell lysates from different developmental stages should be performed. In Leishmania research, for instance, comparing promastigotes cultured at 25°C, heat-shocked promastigotes (37°C for 24h), and axenically cultured amastigotes reveals dramatic differences in CPN10 expression . Western blot quantification should be normalized to total protein loading (verified by Coomassie Blue staining of identical samples) . For more precise quantification, researchers can employ ELISA-based methods or quantitative immunofluorescence with appropriate controls and standards.

How can CPN10 antibodies be applied in comparative studies of stress responses across species?

CPN10 antibodies can be valuable tools for comparative studies of stress responses, particularly heat shock responses, across different species. When designing such experiments, researchers should:

• Verify antibody cross-reactivity with CPN10 from multiple species of interest

• Subject organisms to standardized stress conditions (e.g., heat shock at defined temperatures)

• Prepare standardized lysates for immunoblot analysis

• Quantify relative CPN10 induction compared to housekeeping proteins

• Correlate CPN10 induction patterns with physiological stress responses

It's important to note that in some organisms like Leishmania, CPN10 shows more pronounced induction under heat stress compared to other chaperones such as Hsp70 and Hsp90, which show only marginal increases .

How should researchers address epitope masking when working with CPN10 antibodies?

Epitope masking can occur when CPN10 forms complexes with CPN60 or other proteins, potentially preventing antibody recognition. To address this issue:

• Use multiple antibodies targeting different CPN10 epitopes

• Consider using denaturing conditions for applications like Western blotting

• For native applications, select antibodies raised against peptides known to remain accessible in the native complex

• When analyzing mitochondrial samples, include detergent optimization steps to ensure adequate accessibility

• Compare results from antibodies targeting different regions of CPN10 to identify potential masking effects

Epitope mapping studies have shown that peptides spanning residues 1-76 of CPN10 can produce effective antibodies , suggesting these regions may contain accessible epitopes even in native complexes.

What controls are essential when using CPN10 antibodies for immunofluorescence microscopy?

When performing immunofluorescence microscopy with CPN10 antibodies, researchers should include:

• Pre-immune serum controls to assess background and non-specific binding

• Mitochondrial co-localization markers (e.g., MitoTracker dyes or antibodies against established mitochondrial proteins)

• Competitive blocking with recombinant CPN10 or immunizing peptide

• Secondary antibody-only controls

• Cells with confirmed low or absent CPN10 expression as negative controls

For validation of mitochondrial localization, co-immunoelectron microscopy can be performed using anti-CPN10 antibodies in conjunction with antibodies against established mitochondrial markers .

How can researchers ensure antibody specificity when studying CPN10 in different subcellular fractions?

To ensure specificity when studying CPN10 across different subcellular fractions:

• Validate fractionation procedure using established markers for each compartment:

  • Mitochondria: ATP synthase or cytochrome c

  • Cytosol: GAPDH or lactate dehydrogenase

  • Nucleus: Histone H3 or PCNA

• Perform immunodepletion experiments to confirm signal specificity

• Compare results from polyclonal vs. monoclonal antibodies when available

• Incorporate knockdown/knockout controls where possible

• For organisms with multiple CPN10 isoforms, use peptide-specific antibodies that can distinguish between variants

Research with Leishmania has successfully employed these approaches, confirming that CPN10 localizes specifically to the mitochondrion .

How can CPN10 antibodies help distinguish between stress response pathways in pathogenic organisms?

CPN10 antibodies can be instrumental in characterizing distinct stress response pathways based on differential chaperone induction patterns:

This comparative profile, derived from Leishmania studies , enables researchers to distinguish between general stress responses and stage-specific adaptations. By incorporating CPN10 antibodies into these analyses, researchers can better understand how different pathogens respond to host environments and develop targeted interventions.

What methodological approaches can maximize sensitivity when detecting low-abundance CPN10 expression?

For detecting low-abundance CPN10 expression, researchers should consider:

• Signal amplification techniques:

  • Tyramide signal amplification for immunohistochemistry

  • Chemiluminescent substrates with extended incubation for Western blots

  • Biotin-streptavidin systems for enhanced sensitivity

• Sample preparation optimization:

  • Enrichment of mitochondrial fractions before analysis

  • Use of protease inhibitors to prevent degradation

  • Optimization of extraction buffers for complete solubilization

• Quantitative approaches:

  • Digital imaging with extended exposure times

  • Advanced mass spectrometry for protein identification and quantification

  • Real-time PCR for transcript level analysis as complement to protein detection

In Leishmania promastigotes cultured at 25°C, CPN10 expression is quite low and difficult to detect without optimization , making these considerations particularly relevant.