CPN10 Antibody

What is CPN10 and why is it important in research?

CPN10 (also known as HSP10 or HSPE1) is a ~10 kDa chaperonin protein that plays a fundamental role in protein folding and assembly. It functions as a cochaperone and interacts with members of the HSP60 family to promote proper folding of polypeptides . CPN10 is essential for mitochondrial protein biogenesis and binds to CPN60 in the presence of Mg-ATP, suppressing its ATPase activity . The importance of CPN10 in research stems from its involvement in cellular health and homeostasis, with dysregulation implicated in various diseases including neurodegenerative disorders and cancer . Additionally, human CPN10 has been reported to be identical to Early Pregnancy Factor (EPF), which is involved in control over cell growth and development .

What types of CPN10 antibodies are available for research applications?

Based on current research tools, there are several types of CPN10 antibodies available:

• Monoclonal antibodies:

  • Rabbit monoclonal antibodies (e.g., CAB5580) with reactivity to human, mouse, and rat samples

  • Mouse monoclonal antibodies (e.g., clone M1.2) for specific research applications

• Polyclonal antibodies:

  • Peptide-specific antibodies targeting different epitopes of CPN10, such as:

    • N-terminal sequence (residues 1-11)

    • Internal sequence (residues 33-44)

    • C-terminal peptide (residues 87-101)

    • Full-length CPN10 (residues 1-101)

Each antibody type offers distinct advantages depending on the research application, with monoclonal antibodies providing high specificity and polyclonal antibodies offering broader epitope recognition.

Where is CPN10 localized in cells, and how does this affect antibody selection?

CPN10 is primarily localized in the mitochondrial matrix . This subcellular localization is critical to consider when selecting antibodies for specific applications. For immunofluorescence studies, researchers should select antibodies that have been validated for detecting mitochondrial proteins and consider cell permeabilization protocols that effectively expose mitochondrial epitopes. For western blot applications, proper sample preparation to release mitochondrial proteins is essential. When choosing between antibodies, consider those specifically validated for mitochondrial protein detection and applications that match your experimental requirements (e.g., WB, IHC-P, IF/ICC) . Antibodies targeting different epitopes may show variable efficacy depending on the accessibility of these regions within the mitochondrial environment.

What are the optimal conditions for using CPN10 antibodies in Western blot applications?

For Western blot applications using CPN10 antibodies, researchers should consider the following methodological approach:

• Sample preparation:

  • Include protease inhibitors in lysis buffers to prevent degradation

  • Use mitochondrial enrichment protocols when studying mitochondrial CPN10

  • Consider denaturation conditions carefully as they may affect epitope recognition

• Dilution ratios:

  • For rabbit monoclonal antibodies (e.g., CAB5580): Use at 1:500 - 1:2000 dilution

  • For mouse monoclonal antibodies: Use at approximately 0.25 μg/ml

  • For polyclonal antibodies: Typically 1:2,000 with ECL detection systems

• Detection considerations:

  • Look for bands at the expected molecular weight of ~10-11 kDa

  • Note that some antibodies may detect CPN10 at slightly higher molecular weights (~15 kDa)

  • The most sensitive antibody epitope reported was α77–101, which detected as little as 5 ng antigen (0.5 pmol)

• Controls:

  • Include positive control samples (e.g., MCF7, C6, U-251MG, Neuro-2a, rat kidney)

  • Consider using CPN10 knockout/knockdown samples as negative controls when available

Optimal results require empirical optimization for each specific antibody and sample type.

How can CPN10 antibodies be effectively used in immunohistochemistry and immunofluorescence?

For immunohistochemistry (IHC) and immunofluorescence (IF) applications:

• Tissue/cell preparation:

  • For IHC-P (paraffin-embedded tissues): Proper antigen retrieval is critical; heat-induced epitope retrieval in citrate buffer (pH 6.0) is often effective

  • For IF/ICC: Fixation with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100 typically works well for mitochondrial proteins

• Antibody dilutions:

  • For IHC-P: Use rabbit monoclonal antibodies at 1:100 - 1:500 dilution

  • For IF/ICC: Use antibodies at 1:50 - 1:200 dilution

• Detection systems:

  • For IHC: HRP-conjugated secondary antibodies with DAB substrate

  • For IF: Fluorescently-labeled secondary antibodies compatible with the primary antibody host species

• Colocalization studies:

  • Consider dual staining with established mitochondrial markers (e.g., TOMM20, COX IV)

  • Use confocal microscopy to confirm mitochondrial localization

• Controls:

  • Include peptide competition assays to confirm specificity

  • Use tissues known to express CPN10 at high levels as positive controls

What methods are available for quantifying anti-CPN10 antibody titers in research and clinical samples?

For quantifying anti-CPN10 antibody titers:

• ELISA-based methods:

  • A validated, titer-based ELISA method can be developed using microtitre plates coated with CPN10

  • Serial dilutions of serum samples are added to detect CPN10-specific antibodies

  • Detection using anti-human Ig antibodies conjugated to a detection system

  • A four-fold increase in antibody titer above baseline is typically considered significant, based on:

    • Empirical determination of assay variance (typically up to two-fold in repeat testing)

    • Previously published reports with similar titer-based assays

• Immunoprecipitation methods:

  • Surface proteins on intact cells can be labeled (e.g., iodinated)

  • Cells are lysed and anti-CPN10 antibodies used to precipitate labeled material

  • Specificity can be assessed by comparing with control antibodies (e.g., anti-ovalbumin)

  • Precipitated species can be analyzed by SDS-PAGE to confirm molecular weight

• Western blot quantification:

  • Standard curves can be created using purified recombinant CPN10 at known concentrations

  • Serum or other samples can be analyzed alongside the standard curve

  • Detection limits vary by antibody; some can detect as little as 5 ng antigen

How do different CPN10 antibody epitopes affect experimental outcomes and interpretation?

The choice of antibody epitope significantly impacts experimental outcomes:

• Epitope immunogenicity differences:

  • N-terminal sequence (residues 1-11) and internal sequence (residues 33-44) show poor immunogenicity and declining antibody production even with booster doses

  • C-terminal peptide (residues 87-101) demonstrates better immunogenicity but may not be equally effective in all rabbits

  • Full-length CPN10 (α1-101) generally produces more robust antibody responses

• Technique-specific epitope considerations:

  • For immunoprecipitation: α1-101, α1-28, and α77-101 can detect labeled CPN10, with α1-101 being significantly more efficient

  • For immunoblotting: α77-101 shows the highest sensitivity, detecting as little as 5 ng antigen

• Structural context:

  • Antibodies targeting regions involved in interactions with CPN60/HSP60 may be affected by complex formation

  • Conformational changes in CPN10 during its functional cycle may mask or expose different epitopes

• Experimental interpretation:

  • When different antibodies targeting distinct epitopes yield conflicting results, consider:

    • Post-translational modifications that might affect epitope recognition

    • Protein interactions that could mask specific epitopes

    • Potential isoforms or proteolytic fragments that contain only certain epitopes

What is known about CPN10's role in cell differentiation, and how can CPN10 antibodies help elucidate these mechanisms?

CPN10 has been identified as an endothelial-derived differentiation factor with broader implications:

• Observed differentiation effects:

  • Decreases cell proliferation in K562 erythroleukemia cells

  • Increases erythroid differentiation markers (glycophorin A, hemoglobin) in TF-1 cells

  • Enhances collagen I expression in skin fibroblasts overexpressing CPN10

• Signaling mechanisms:

  • Early changes in K562 cells after CPN10 treatment include various phosphorylation events

  • Decreases GSK-3alpha phosphorylation

  • Decreases cofilin-1 phosphorylation while stimulating GSK-3beta phosphorylation

  • GSK-3-regulated pathways appear important for differentiation, as glycophorin A production decreases with GSK-3 inhibition

• Research approaches using CPN10 antibodies:

  • Western blot analysis to monitor changes in CPN10 expression during differentiation

  • Immunoprecipitation to identify interaction partners during differentiation processes

  • Immunofluorescence to track subcellular localization changes during differentiation

  • Neutralization experiments to block extracellular CPN10 effects

  • ChIP assays to investigate potential transcriptional regulatory roles

• Experimental design considerations:

  • Include appropriate time-course analyses to capture both early and late differentiation events

  • Combine antibody-based detection with functional assays for differentiation markers

  • Consider the interplay between mitochondrial and extracellular CPN10

How can CPN10 antibodies be used to investigate the relationship between CPN10 and immune modulation?

CPN10 has demonstrated immune-modulatory properties that can be investigated using antibodies:

• Cytokine production assessment:

  • CPN10 treatment has been shown to affect cytokine production:

    • Significant reduction in TNF-α and IL-1β levels by week 8 of treatment

    • Reduction in IL-8 and IL-10 levels by week 8, returning to baseline by week 12

    • No observed changes in IL-6 and IL-12 levels

• Experimental approaches:

  • Isolate PBMCs and stimulate with LPS to measure cytokine production

  • Use flow cytometry or cytometric bead array technology to quantify cytokine levels

  • Compare cytokine profiles before and after CPN10 treatment

  • Use neutralizing CPN10 antibodies to block effects and confirm specificity

• Anti-CPN10 antibody monitoring:

  • Develop ELISA-based methods to detect anti-CPN10 antibodies in patient samples

  • A four-fold increase in antibody titer above baseline is considered significant

  • Monitor antibody development during CPN10 treatment (found in 8% of treated subjects)

• Imaging approaches:

  • Use immunohistochemistry to evaluate CPN10 expression in immune tissues

  • Perform co-localization studies with immune cell markers

What are the common challenges when using CPN10 antibodies and how can they be addressed?

Researchers face several challenges when working with CPN10 antibodies:

• Molecular weight variations:

  • Expected molecular weight is ~10-11 kDa, but some antibodies detect bands at ~15 kDa

  • Solution: Verify specificity using positive controls and consider post-translational modifications or oligomerization that might affect migration

• Cross-reactivity concerns:

  • CPN10 shares structural similarities with other small heat shock proteins

  • Solution: Perform peptide competition assays and include appropriate negative controls

• Low signal in Western blots:

  • CPN10 is relatively small and may transfer inefficiently

  • Solution: Use PVDF membranes with small pore sizes, optimize transfer conditions (time, voltage, buffer composition), and consider using more sensitive detection methods

• Subcellular localization challenges:

  • Mitochondrial localization requires effective permeabilization

  • Solution: Test different fixation and permeabilization protocols; use mitochondrial markers as controls

• Variable immunogenicity:

  • Different epitopes show varying immunogenicity profiles

  • Solution: When generating custom antibodies, consider using full-length protein or highly immunogenic epitopes (e.g., C-terminal region)

How do you differentiate between specific and non-specific signals when using CPN10 antibodies?

To distinguish between specific and non-specific signals:

• Validation controls:

  • Positive controls: Use samples known to express CPN10 (e.g., MCF7, C6, U-251MG, Neuro-2a, rat kidney)

  • Negative controls: Use CPN10 knockout/knockdown samples, or tissues/cells with negligible CPN10 expression

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

• Technical approaches:

  • Antibody titration: Test a range of dilutions to find optimal signal-to-noise ratio

  • Secondary antibody-only controls: Omit primary antibody to identify non-specific secondary antibody binding

  • Isotype controls: Use irrelevant antibodies of the same isotype and host species

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes

  • Consistent results across different antibodies increase confidence in specificity

• Functional validation:

  • Verify that observed changes correlate with known CPN10 biology

  • Confirm findings using complementary techniques (e.g., mass spectrometry)

How can researchers interpret conflicting data when using different anti-CPN10 antibodies?

When faced with conflicting results using different anti-CPN10 antibodies:

• Epitope accessibility analysis:

  • Map the epitopes of each antibody to the CPN10 structure

  • Consider whether protein interactions or conformational changes might affect epitope recognition

  • Evaluate whether specific epitopes might be masked in certain experimental conditions

• Post-translational modification considerations:

  • Determine if epitopes contain potential sites for post-translational modifications

  • Test whether treatments affecting modifications (e.g., phosphatase treatment) resolve discrepancies

• Isoform detection:

  • Investigate whether antibodies might be detecting different isoforms or processed forms

  • Use RT-PCR to assess transcript variants

  • Consider complementary techniques (e.g., mass spectrometry) to identify exact protein species

• Methodological approach:

  • Compare antibody performance across different applications (WB, IHC, IP)

  • Test different sample preparation methods to see if they resolve discrepancies

  • Consider antibody batch variation and storage conditions

• Validation strategy:

  • Prioritize data from antibodies with most extensive validation

  • Use genetic approaches (overexpression, knockdown) to confirm specificity

  • Consider developing new antibodies against conserved epitopes

How are CPN10 antibodies being used to investigate its role in disease pathology?

CPN10 antibodies are enabling various approaches to understand disease connections:

• Neurodegenerative disorders:

  • Immunohistochemical analysis of CPN10 expression in patient tissues

  • Correlation of CPN10 levels with disease progression

  • Investigation of protein misfolding stress responses involving CPN10

• Cancer research:

  • Analysis of CPN10 expression changes across cancer types and stages

  • Correlation with patient outcomes and therapeutic responses

  • Investigation of mitochondrial dysfunction in cancer involving CPN10

• Inflammatory conditions:

  • Monitoring changes in CPN10 levels during inflammation

  • Tracking immune cell responses to CPN10 using flow cytometry

  • Investigating CPN10's impact on inflammatory cytokine production

• Multiple sclerosis research:

  • Evaluating CPN10 as a potential therapeutic

  • Monitoring anti-CPN10 antibody development during treatment

  • Assessing effects on cytokine profiles and MRI lesion development

• Reproductive biology:

  • Investigating the relationship between CPN10 and Early Pregnancy Factor

  • Studying roles in early embryonic development

  • Analyzing maternal serum levels and correlation with pregnancy outcomes

What novel methodologies are being developed for studying CPN10 interactions using antibody-based approaches?

Emerging methodologies for studying CPN10 interactions include:

• Proximity ligation assays (PLA):

  • Detect CPN10 interactions with CPN60/HSP60 and other partners in situ

  • Visualize interactions within specific cellular compartments

  • Quantify changes in interaction patterns under different conditions

• BioID or APEX proximity labeling:

  • Fuse CPN10 to biotin ligase (BioID) or peroxidase (APEX)

  • Identify proximal proteins that may interact transiently

  • Use CPN10 antibodies to confirm expression of fusion proteins

• Single-molecule imaging:

  • Combine with fluorescently labeled antibody fragments

  • Track CPN10 dynamics in living cells

  • Analyze co-movement with potential interaction partners

• Mass spectrometry-coupled immunoprecipitation:

  • Use CPN10 antibodies for immunoprecipitation

  • Identify interaction partners by mass spectrometry

  • Quantify changes in interactome under different conditions

• Antibody-based biosensors:

  • Develop FRET-based sensors using antibody fragments

  • Monitor conformational changes or post-translational modifications

  • Assess real-time changes in CPN10 function

How can researchers best integrate CPN10 antibody data with other omics approaches for comprehensive pathway analysis?

Integrating CPN10 antibody data with multi-omics approaches:

• Transcriptomics integration:

  • Correlate CPN10 protein levels (via antibody detection) with mRNA expression

  • Identify conditions where post-transcriptional regulation may be important

  • Analyze transcriptional responses to CPN10 modulation

• Proteomics complementation:

  • Use antibody-based enrichment prior to mass spectrometry analysis

  • Validate proteomics findings with targeted antibody approaches

  • Analyze post-translational modifications detected in proteomics using modification-specific antibodies

• Metabolomics connections:

  • Correlate CPN10 levels with changes in mitochondrial metabolites

  • Investigate metabolic consequences of CPN10 knockdown/overexpression

  • Integrate metabolic flux data with CPN10 localization and interaction data

• Pathway analysis strategies:

  • Use antibody data to establish presence and relative abundance of CPN10

  • Employ computational approaches to integrate antibody-derived data with other omics datasets

  • Develop network models incorporating CPN10 interactions and downstream effects

• Single-cell approaches:

  • Combine CPN10 antibody-based detection with single-cell transcriptomics

  • Analyze cell-to-cell variability in CPN10 expression and correlation with phenotypes

  • Perform trajectory analyses to understand dynamics during cellular processes