CPN10-1 Antibody

What is Cpn10 and where is it expressed?

Cpn10 (Chaperonin 10) is a small heat shock protein with an approximate molecular mass of 10 kDa that functions as a co-chaperone and interacts with members of the Hsp60 family to promote proper folding of polypeptides . The E. coli homolog is known as GroES. Cpn10 acts as a cochaperone with Cpn60 (GroEL in E. coli) to form a functional complex with sevenfold symmetry that assists in protein folding and assembly processes .

Interestingly, Cpn10 has been identified as Early Pregnancy Factor (EPF), an extracellular molecule with growth-regulatory and immunomodulatory properties. This dual identity raises unprecedented regulatory and mechanistic questions about its various biological roles . In human cells, Cpn10 is primarily localized in the mitochondrial matrix, but immunohistochemical studies have shown that in cancer cells, it can also display intense apical membrane staining and appear within secreted material in malignant gland spaces, suggesting either active secretion or release .

What types of Cpn10 antibodies are available for research?

Several types of antibodies against Cpn10 are available for research applications:

Monoclonal antibodies:

• Mouse monoclonal antibodies (e.g., clone M1.2) useful for EIA, IP, and Western blotting applications, with a typical detection sensitivity of about 0.25 μg/ml .

Polyclonal antibodies:

• Rabbit polyclonal antibodies targeting specific regions of the Cpn10 protein:

  • N-terminal sequence (residues 1-11, 1-28)

  • Internal sequences (residues 29-56, 33-44, 57-76)

  • C-terminal sequence (residues 77-101, 87-101)

  • Full-length recombinant Cpn10 (residues 1-101)

Each antibody exhibits different properties regarding specificity, sensitivity, and preferred applications. For example, antibodies against residues 77-101 have shown excellent performance in immunoblotting, while those against acetylated N-terminus (residues 1-28) have demonstrated outstanding results in immunohistochemistry .

What are the optimal applications for different Cpn10 antibodies?

Different Cpn10 antibodies show varying performance across applications, and choosing the right antibody is critical for experimental success:

Western Blotting:

• C-terminal targeted antibodies (α77-101) have demonstrated high sensitivity, detecting as little as 5 ng (0.5 pmol) of antigen .

• These antibodies may preferentially recognize oligomeric forms of Cpn10, which is an important consideration for data interpretation .

• Expected band size is approximately 10 kDa, though some detection systems may show bands closer to 15 kDa .

Immunohistochemistry:

• Antibodies targeting the N-terminal region with acetylated terminus (αAc1-28) have shown outstanding properties for IHC applications .

• These antibodies can effectively detect Cpn10 in paraffin-embedded tissues such as lung carcinoma and colonic tissue .

Immunofluorescence:

• Several antibodies work well for cellular localization studies, showing the typical punctate mitochondrial pattern .

• Dilutions between 1:100 to 1:200 have been effective for immunofluorescent detection in fixed cells .

ELISA:

• Sandwich ELISA using combinations like α77-101 with α29-56 or α57-76, αAc1-28 with α29-56, and anti-whole molecule α1-101 with itself have shown the highest sensitivity .

• The current detection limit is approximately 500 pM, which may not be sufficient for detecting physiological levels in serum .

Immunoprecipitation:

• Antibodies recognizing the whole molecule (α1-101) are considerably more efficient for immunoprecipitation than epitope-specific antibodies .

• Pre-clearing steps with irrelevant antibodies can significantly reduce non-specific background in immunoprecipitation experiments .

How should I optimize Western blot protocols for Cpn10 detection?

Optimizing Western blot protocols for Cpn10 requires consideration of several factors:

Sample preparation considerations:

• Cpn10 naturally forms heptamers that can persist even under denaturing conditions, resulting in detection of monomers (~10 kDa), dimers (~20 kDa), and higher-order oligomers .

• To enhance denaturation and focus on monomeric forms, consider:

  • Extended boiling times in sample buffer (5+ minutes)

  • Higher SDS concentrations (up to 2%)

  • Additional reducing agents

  • Urea addition (up to 8M) for particularly stubborn samples

Gel and transfer parameters:

• Use higher percentage gels (15-18%) for better resolution of the small Cpn10 protein

• Consider gradient gels (4-20%) if detecting both monomeric and oligomeric forms

• Transfer to PVDF membranes has been successfully demonstrated

• Use lower molecular weight transfer conditions (higher methanol percentage, shorter transfer times)

Antibody selection and concentration:

• For highest sensitivity, antibodies against residues 77-101 have demonstrated excellent performance

• Start with recommended dilutions (e.g., 0.25 μg/ml) and optimize as needed

• Be aware that some antibodies may preferentially detect certain forms (monomeric vs. oligomeric)

Controls:

• Include purified recombinant Cpn10 as a positive control

• Consider molecular weight markers optimized for low molecular weight proteins

• Include a lysate from cells with Cpn10 knockdown if available for specificity verification

Visualization:

• Enhanced chemiluminescence detection has been successfully used

• For quantitative analysis, consider fluorescent secondary antibodies and appropriate imaging systems

What are the best fixation methods for immunolocalization of Cpn10?

Proper fixation is critical for accurate immunolocalization of Cpn10:

Cell culture immunofluorescence:

• 4% paraformaldehyde fixation for 15 minutes at room temperature has been successfully used for cell lines including U2OS and HeLa .

• This fixation method preserves the typical punctate mitochondrial staining pattern of Cpn10 .

• For co-localization studies, counterstaining with cytoskeletal markers (alpha-tubulin) and nuclear stains (Hoechst 33342) can provide excellent contextual information .

Tissue immunohistochemistry:

• Paraffin embedding following appropriate fixation has yielded good results for detecting Cpn10 in tissues such as human lung carcinoma and colonic tissue .

• Antigen retrieval may be necessary for paraffin sections to expose epitopes masked during fixation.

• For IHC applications, the antibody targeting acetylated N-terminus (αAc1-28) has shown outstanding performance .

Expected staining patterns:

• In normal cells: Primarily punctate cytoplasmic staining consistent with mitochondrial localization .

• In cancer cells: Additional patterns may include intense apical membrane staining and presence within secreted material in gland spaces, suggesting potential secretion or release of Cpn10 .

Troubleshooting poor staining:

• If mitochondrial staining is weak, consider gentle permeabilization methods to preserve mitochondrial integrity while allowing antibody access.

• For detecting extracellular/secreted Cpn10, minimize permeabilization to reduce background from intracellular pools.

• Co-staining with established mitochondrial markers can help confirm proper fixation and permeabilization conditions.

How can I validate the specificity of my Cpn10 antibody?

Thorough validation of antibody specificity is essential for reliable research results:

Western blot validation:

• Look for a single band at the expected molecular weight (~10 kDa for monomer) .

• Compare with purified recombinant Cpn10 as a positive control.

• When possible, include Cpn10 knockout/knockdown samples as negative controls.

• Be aware that under some conditions, oligomeric forms may persist, resulting in additional bands at higher molecular weights .

Peptide competition assay:

• Pre-incubate the antibody with excess immunizing peptide before application.

• This should abolish or significantly reduce specific signal while leaving non-specific binding unaffected.

• Particularly useful for polyclonal antibodies raised against specific peptide epitopes .

Multiple antibody approach:

• Use antibodies targeting different epitopes of Cpn10 (e.g., N-terminal and C-terminal).

• Consistent results with multiple antibodies significantly increase confidence in specificity.

• This approach can also help identify any conformation-dependent recognition issues .

Immunoprecipitation-based validation:

• Use the antibody to immunoprecipitate from cell lysates.

• Analyze the precipitated material by mass spectrometry to confirm Cpn10 identity.

• Some antibodies like α1-101 have demonstrated precise molecular recognition patterns in immunoprecipitation experiments .

Functional validation:

• For antibodies claiming to neutralize EPF activity, functional assays can confirm specificity.

• Previous studies found that antibodies against residues 1-76 produced EPF-neutralizing activity, while those against residues 77-101 did not .

How can I distinguish between monomeric and oligomeric forms of Cpn10?

Distinguishing between different oligomeric states of Cpn10 presents unique challenges:

Antibody selection strategies:

• Some antibodies show preferential recognition of different oligomeric states. For example, antibodies against residues 77-101 have been observed to preferentially recognize oligomeric forms in immunoblots .

• This region (77-101) appears to be directed away from subunit binding interfaces, while residues 1-76 may be involved in binding interactions .

• Using multiple antibodies targeting different epitopes can help identify the predominant forms in your samples.

Gel electrophoresis approaches:

• Native PAGE preserves the native heptameric state and can be followed by Western blotting.

• Blue native PAGE with mild detergents can help resolve different oligomeric states.

• For SDS-PAGE, modulating the denaturation conditions can shift the equilibrium between monomers and oligomers:

  • Stronger denaturation (higher temperatures, SDS concentrations) favors monomers

  • Milder conditions may preserve some oligomeric forms

Size exclusion chromatography combined with antibody detection:

• SEC can separate monomeric (~10 kDa) from heptameric (~70 kDa) Cpn10

• Following separation, Western blotting with appropriate antibodies can confirm the identity of each fraction

Structural data visualization:

• Even under denaturing SDS-PAGE conditions, dimeric and higher-order oligomeric forms can persist and be visualized with very sensitive detection methods .

• Heavily exposed blots may reveal higher-order oligomers beyond the typical monomer and dimer bands .

Functional implications:

• When interpreting results, consider that Cpn10 functions as a non-covalent heptamer in vivo .

• The differential recognition of oligomeric states by different antibodies may reflect important structural characteristics relevant to Cpn10's dual functionality as a chaperonin and as EPF.

What are the challenges in detecting extracellular vs. mitochondrial Cpn10?

Cpn10's dual identity as both a mitochondrial chaperonin and extracellular EPF presents unique detection challenges:

Abundance differences:

• Mitochondrial Cpn10 is typically more abundant than extracellular Cpn10/EPF.

• Current antibody-based assays have detection limits around 500 pM, which may not be sensitive enough for detecting physiological levels of extracellular Cpn10/EPF in serum .

• Higher-affinity antibodies (Kₐ > 10⁷ M⁻¹) would be needed to achieve the low pM detection range typical for cytokines and growth factors .

Selective isolation strategies:

• For mitochondrial Cpn10: Subcellular fractionation focusing on mitochondrial isolation

• For extracellular Cpn10: Analysis of cell culture supernatants or biological fluids

• Critical controls to ensure mitochondrial integrity during fractionation are essential to prevent false detection of "extracellular" Cpn10 from damaged mitochondria

Potential conformational or modification differences:

• Extracellular Cpn10/EPF may undergo post-translational modifications or conformational changes not present on mitochondrial Cpn10.

• Immunohistochemical studies of cancer tissues have shown both mitochondrial staining and potential evidence of secretion, with intense apical membrane staining and presence within secreted material in gland spaces .

Specific detection approaches:

• For fixed samples: Minimal permeabilization can help focus on extracellular/membrane-associated pools

• For biochemical analyses: Selective surface protein labeling (e.g., cell-impermeable biotinylation) followed by Cpn10 immunoprecipitation can identify surface-associated Cpn10

Functional validation:

• EPF bioassays combined with neutralizing antibodies provide functional confirmation of extracellular Cpn10

• Studies have shown that while human platelet-derived EPF and rat mitochondrial Cpn10 were functionally interchangeable in vitro, E. coli GroES did not exhibit activity in the EPF bioassay .

How can I develop improved detection methods for low-abundance Cpn10?

Current antibody-based assays for Cpn10 have limitations in sensitivity that may be addressed through several approaches:

Sandwich ELISA optimization:

• The most sensitive current ELISA combinations (antipeptide antibodies α77-101 with either α29-56 or α57-76, αAc1-28 with α29-56, and anti-whole molecule α1-101 with self) have detection limits of approximately 500 pM .

• This sensitivity is insufficient for detecting physiological levels of EPF in serum .

Higher-affinity antibody development:

• Current anti-Cpn10 antibodies have moderate to high affinity (Kₐ ≈ 10⁷ M⁻¹) .

• To detect low pM concentrations, antibodies with several orders of magnitude higher affinity would be required .

• Historical challenges in generating high-affinity antibodies against Cpn10 suggest novel approaches may be needed :

  • Phage display technology for antibody selection

  • Affinity maturation of existing antibodies

  • Alternative immunization strategies

Signal amplification technologies:

• Polymerized enzyme detection systems

• Tyramide signal amplification for immunohistochemistry

• Digital ELISA platforms with single-molecule detection capabilities

Enrichment strategies:

• Immunoprecipitation followed by Western blotting

• Aptamer-based capture with antibody detection

• Selective isolation of extracellular vesicles that may contain Cpn10

Alternative detection platforms:

• Mass spectrometry-based approaches (MRM/PRM) for targeted detection

• Proximity ligation assays for sensitive in situ detection

• Bioluminescence resonance energy transfer (BRET) systems for real-time monitoring

Technical considerations:

• Sample handling to minimize Cpn10 degradation

• Blocking agents that reduce background without interfering with low-abundance detection

• Optimization of antibody pairs for reduced steric hindrance and improved capture efficiency

How can Cpn10 antibodies support research into novel scaffold applications?

Cpn10 has emerging applications as a scaffold for therapeutic peptide presentation, with antibodies playing a crucial role in developing and characterizing these novel constructs:

Heptameric display platform characterization:

• Human Cpn10 (hCpn10) naturally forms heptamers, providing a multivalent display platform for therapeutic peptides .

• Antibodies against the scaffold portion can help characterize the quaternary structure and stability of these constructs.

• This approach has shown promise, as demonstrated with the Factor VIIa-targeting peptide (E-76) displayed on hCpn10, which showed enhanced anticoagulant activity compared to the free peptide .

Optimization of peptide presentation:

• Molecular dynamics simulations and protein sizing analyses have identified optimal peptide linkers (such as P1) for maintaining the quaternary hCpn10 heptamer structure while displaying functional peptides .

• Antibodies can be used to confirm proper folding and epitope presentation in these engineered constructs.

Functional verification:

• When peptides are displayed on the Cpn10 scaffold, antibodies can help verify that:

  • The scaffold maintains its heptameric structure

  • The displayed peptides maintain their functional conformation

  • The construct exhibits improved functional properties compared to free peptides

• For example, CE76-P1 (a Cpn10 scaffold displaying the Factor VIIa-targeting peptide) showed nanomolar affinity for Factor VIIa (3 nM) similar to the free E-76 peptide (6 nM) but demonstrated enhanced inhibition of coagulation .

Structural data collection:

• Antibodies can be used in combination with structural biology techniques to elucidate the conformation of peptides displayed on the Cpn10 scaffold.

• This information is valuable for rational design of improved scaffolds with optimal linker lengths and geometries.

Comparison with other scaffold platforms:

• Cpn10 joins other non-antibody scaffolds like Affibody and DARPin in the development of alternative binding reagents .

• Antibodies specific to each scaffold type can help researchers compare stability, expression, and functional properties across different platforms.

What are the key considerations when studying Cpn10's role in disease processes?

Cpn10 antibodies can facilitate research into its roles in various diseases, with several important considerations:

Cancer research applications:

• Immunohistochemical studies have shown distinctive Cpn10 staining patterns in cancer cells, including punctate cytoplasmic staining and occasionally intense apical membrane staining .

• The presence of Cpn10 within secreted material in malignant gland spaces suggests potential active secretion or release, which may have functional significance .

• Quantitative analysis of Cpn10 levels in different cancer types might identify potential diagnostic or prognostic biomarkers.

Technical considerations for disease tissue analysis:

• Use antibodies validated for the specific application (e.g., αAc1-28 for immunohistochemistry) .

• Include appropriate controls (normal adjacent tissue, benign pathologies).

• Consider dual staining with markers of cellular compartments to assess localization changes in disease states.

Inflammatory and autoimmune research:

• Given Cpn10's potential immunomodulatory properties, carefully select antibodies that don't interfere with the specific functions being studied.

• For functional studies, consider both neutralizing and non-neutralizing antibodies to distinguish between correlation and causation.

Stress response analysis:

• As a heat shock protein, Cpn10 levels may change in response to cellular stress.

• When analyzing stress-induced changes, consider both total protein levels and subcellular distribution.

• Time-course studies with appropriate antibodies can track the dynamics of these responses.

Reproductive biology applications:

• Given Cpn10's identity as EPF, select antibodies appropriate for detecting its extracellular presence in reproductive tissues and fluids.

• Functional studies may benefit from EPF-neutralizing antibodies, which have been found to target the N-terminal three-quarters of the molecule (residues 1-76) .

How can I utilize epitope-specific Cpn10 antibodies to understand structure-function relationships?

Strategic use of antibodies targeting different epitopes can provide insights into Cpn10's structure-function relationships:

Functional domain mapping:

• Antibodies against residues 1-76 produce EPF-neutralizing activity, while those against residues 77-101 do not, suggesting the N-terminal three-quarters of the molecule is involved in binding to lymphocytes .

• This pattern indicates that the C-terminal quarter of the molecule is directed away from the binding site .

• By testing the effects of different epitope-specific antibodies on various Cpn10 functions, researchers can map functional domains without requiring protein engineering.

Oligomerization interface analysis:

• Some antibodies may preferentially recognize oligomeric or monomeric forms, providing insights into accessible epitopes in each state.

• The observation that antibodies to residues 77-101 preferentially recognize oligomeric forms suggests this region may be more exposed in the assembled heptamer .

Conformational change detection:

• Comparing binding patterns of different epitope-specific antibodies under various conditions can reveal conformational changes.

• This approach can help elucidate how Cpn10 might adopt different conformations in its dual roles as a chaperonin and as EPF.

Experimental design considerations:

• Use multiple antibodies targeting different epitopes in parallel experiments.

• Compare results across different techniques (e.g., ELISA, immunoprecipitation, immunohistochemistry) as certain epitopes may be preferentially exposed in different sample preparation methods.

• Consider how antibody binding might itself affect Cpn10 structure or function when interpreting results.

Application to therapeutic development:

• Understanding which epitopes are critical for specific functions can guide the development of function-selective inhibitors or activators.

• For therapeutic applications of Cpn10 itself, epitope mapping can identify regions that should be preserved or modified to enhance desired activities.

Why might I observe multiple bands when probing for Cpn10?

Observing multiple bands in Cpn10 Western blots is common and can occur for several reasons:

Oligomeric states:

• Cpn10 functions as a heptamer, and even under denaturing SDS-PAGE conditions, dimeric and higher-order oligomeric forms can persist .

• This is evidenced by the observation of dimers and higher oligomers on heavily exposed gels, particularly when using the α77-101 antibody .

• Expected band patterns may include:

  • Monomer: ~10 kDa (primary band)

  • Dimer: ~20 kDa

  • Higher oligomers: Various sizes above 20 kDa

Sample preparation effects:

• Insufficient denaturation can preserve oligomeric forms

• Boiling time, SDS concentration, and reducing agent concentration all affect the monomer/oligomer ratio

• Some antibodies, particularly those targeting residues 77-101, may preferentially recognize oligomeric forms

Post-translational modifications:

• Modifications such as phosphorylation, acetylation, or ubiquitination can cause shifts in apparent molecular weight

• N-terminal acetylation of Cpn10 is one such known modification that can affect antibody recognition

Precursor forms:

• As a mitochondrial protein, Cpn10 may be detected in both its mature form and as a precursor containing a mitochondrial targeting sequence

Validation approaches:

• Compare with purified recombinant Cpn10 positive control

• Use multiple antibodies targeting different epitopes to confirm band identity

• Consider native PAGE to better preserve and visualize the native heptameric state

• Pre-clearing steps with irrelevant antibodies can help reduce non-specific background in complex samples

What are the best practices for optimizing Cpn10 immunoprecipitation?

Successful immunoprecipitation of Cpn10 requires careful attention to several factors:

Antibody selection:

• Antibodies recognizing the whole molecule (α1-101) have shown considerably greater efficiency for immunoprecipitation than epitope-specific antibodies .

• Some epitope-specific antibodies (α1-28, α77-101) can also perform well but may be less efficient .

• For surface-associated Cpn10, ensure the antibody can recognize native, non-denatured protein.

Pre-clearing strategy:

• Pre-clearing with irrelevant antibodies significantly reduces non-specific background .

• This is particularly evident when comparing the precipitation patterns of anti-Cpn10 antibodies with control antibodies like anti-ovalbumin .

Buffer optimization:

• For mitochondrial Cpn10: Consider gentle lysis buffers that preserve protein-protein interactions if studying Cpn10-Cpn60 complexes

• For extracellular Cpn10: Use buffers optimized for secreted proteins

• Buffer composition can significantly affect the oligomeric state of Cpn10

Detection techniques:

• Western blotting of immunoprecipitated material can confirm the identity of precipitated proteins .

• For enhanced sensitivity, consider protein labeling approaches (e.g., iodination of cell surface proteins prior to lysis and immunoprecipitation) .

Controls:

• Include isotype-matched irrelevant antibodies as negative controls

• Perform parallel immunoprecipitations with antibodies targeting different Cpn10 epitopes

• Consider pre-incubation with immunizing peptides to demonstrate specificity

Verification methods:

• Compare the migration of immunoprecipitated material with pure Cpn10 to confirm identity .

• For further validation, consider mass spectrometry analysis of immunoprecipitated bands.

How can I address inconsistent results between different anti-Cpn10 antibodies?

Inconsistencies between different anti-Cpn10 antibodies are common and understanding their causes can help with experimental design and data interpretation:

Epitope-specific recognition patterns:

• Different antibodies target distinct epitopes that may be differentially accessible depending on Cpn10's conformation, oligomeric state, or interactions .

• For example, antibodies against residues 77-101 appear to preferentially recognize oligomeric forms, while others may more accurately reflect the monomeric:oligomeric ratio .

Systematic comparison approach:

• Test multiple antibodies side-by-side under identical conditions

• Document their performance across different applications (WB, IHC, IP, etc.)

• Create a reference table similar to the characterization done in previous studies

Application-specific optimization:

• Some antibodies perform better in certain applications; for instance:

  • α77-101 shows excellent performance in immunoblotting

  • αAc1-28 demonstrates outstanding properties in immunohistochemistry

  • α1-101 (anti-whole molecule) is considerably more efficient for immunoprecipitation

Technical considerations:

• Fixation methods may affect epitope accessibility differently for different antibodies

• Sample preparation (native vs. denatured) can dramatically change which antibody works best

• Some antibodies (like α29-56 and α57-76) work effectively only as detection antibodies and not as capture antibodies in sandwich ELISAs

Validation strategies:

• Use peptide competition assays specific to each antibody's target epitope

• Employ genetic approaches (knockout/knockdown) to confirm specificity

• Consider alternative detection methods to corroborate antibody-based findings

Data interpretation guidelines:

• When results differ between antibodies, consider which epitopes they target and how this might relate to Cpn10's structure and function

• Differences may reveal biologically relevant information about Cpn10's state rather than simply representing technical issues

• When reporting results, clearly specify which antibody was used and its target epitope