CAPN10 Antibody, Biotin conjugated

What is CAPN10 and why is it an important research target?

CAPN10 (Calpain-10) is a calcium-regulated non-lysosomal thiol-protease that catalyzes limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction. It was identified as the first candidate susceptibility gene for type 2 diabetes mellitus (T2DM) . CAPN10 belongs to the calpain family but is considered atypical as it lacks the calmodulin-like calcium-binding domain found in typical calpain members, instead having a divergent C-terminal domain .

The significance of CAPN10 in research stems from its role in multiple cellular processes. It processes microtubule associated protein 1 (MAP1) family proteins into heavy and light chains and regulates their binding activities to microtubules and actin filaments . This functionality is critical for proper actin dynamics and cytoskeletal organization. Additionally, studies with pancreatic islets from CAPN10 knockout mice demonstrated significantly increased insulin secretion at both high and low glucose levels, suggesting CAPN10 deficiency affects insulin secretion through abnormal actin reorganization .

What are the functional characteristics of CAPN10 that make it unique among calpain family members?

CAPN10 possesses several distinctive characteristics that differentiate it from other calpain family members:

• Calcium-independence: Unlike typical calpain members that require calcium for proteolytic activity, CAPN10 exhibits proteolytic activity both with and without Ca²⁺. In vitro assays show CAPN10 is capable of cleaving MAP1B even in the presence of 5 mM EDTA, confirming it does not require Ca²⁺ or other metal ions for its proteolytic activity .

• Structural uniqueness: CAPN10 lacks the calmodulin-like calcium-binding domain (domain IV) that is well conserved among typical calpain members. Instead, it has a divergent C-terminal domain, making it structurally similar to calpains 5 and 6 .

• Substrate specificity: CAPN10 specifically processes MAP1 family proteins (MAP1A, MAP1B, and MAP1S) into heavy and light chains, regulating their binding activities to microtubules and actin filaments .

• Role in cytoskeletal dynamics: CAPN10 regulates actin dynamics through MAP1B cleavage. When CAPN10 is knocked down or knocked out, there is increased formation of thick actin stress fibers and reduced actin dynamics, as demonstrated by fluorescence recovery after photo-bleaching (FRAP) analysis .

These unique characteristics make CAPN10 a particularly interesting target for research into cytoskeletal regulation and metabolic disorders.

How do CAPN10 gene polymorphisms affect susceptibility to type 2 diabetes?

CAPN10 gene polymorphisms have been associated with susceptibility to type 2 diabetes in multiple studies, though the relationship varies by population:

A study in the Korean population identified a novel 111/121 diplotype (SNP-43, -19, and -63) associated with a high risk of T2DM with an odds ratio of 2.580 (95% confidence interval = 1.602-4.155, P = 0.001) . Interestingly, this differed from the high-risk haplotype (112/121) identified in Mexican-Americans, which was not significant in the Korean population .

Population genetics studies of CAPN10 have revealed two distinct deviations from the standard neutral model:

• A significant deficit of variation in the haplotype class defined by the derived allele at SNP44, suggesting this haplotype was quickly driven to high frequency by positive natural selection. Interestingly, the derived allele at SNP44 is protective against diabetes .

• A local excess of polymorphism and linkage disequilibrium decay in intron 13, potentially explained by long-standing balancing selection that maintains multiple selected alleles .

These findings suggest that different CAPN10 genetic variants may have been subject to different selective pressures across human evolution, possibly related to energy metabolism and storage efficiency (the "thrifty genotype" hypothesis).

What are the optimal experimental conditions for using biotin-conjugated CAPN10 antibodies?

When working with biotin-conjugated CAPN10 antibodies, researchers should consider the following experimental conditions for optimal results:

Storage conditions:

• Store at -20°C for up to 1 year from the date of receipt

• Avoid repeated freeze-thaw cycles

• For short-term storage, some products can be kept at 4°C for up to one month

Buffer compatibility:

• Most biotin-conjugated antibodies are supplied in PBS containing components such as:

  • 50% glycerol

  • 0.5% BSA

  • 0.02% sodium azide

• For conjugation reactions, use Hepes, MES, MOPS, or other amine-free buffers. Tris buffer can be used up to 20mM with minimal reduction in coupling efficiency

Antibody concentration:

• For western blotting, the recommended dilution range for CAPN10 antibodies is typically 1:500-1:2000

• For ELISA applications, dilutions may be much higher (e.g., 1:40000)

Sample preparation:

• Ensure purification of antibodies before conjugation to avoid labeling of contaminant proteins

• Avoid ascites fluid, serum, or hybridoma culture media for conjugation procedures

• For optimal conjugation results, antibody concentrations of 1-4 mg/ml are generally recommended

Detection systems:

• For biotin-conjugated antibodies, streptavidin-labeled molecules (such as Streptavidin-HRP) are used for detection

• In ELISA applications, the biotin-conjugated CAPN10 antibody serves as a detection antibody in a sandwich format

What methodological considerations are important when using biotin-conjugated CAPN10 antibodies in proximity labeling experiments?

Proximity labeling using biotin-conjugated antibodies represents an innovative approach for identifying protein interactions in fixed cells and primary tissues. When using biotin-conjugated CAPN10 antibodies for proximity labeling, consider these methodological aspects:

Biotinylation by Antibody Recognition (BAR) technique:

• BAR is a novel, proximity-based labeling approach that uses antibodies to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues

• Unlike traditional proximity labeling methods that require fusion gene insertion, BAR does not require genetic manipulation, making it suitable for primary human tissue samples

Protocol considerations:

• Fixation: Begin with proper fixation of cells or tissue samples to preserve cellular architecture while maintaining protein antigenicity

• Primary antibody binding: Use anti-CAPN10 antibody to target the protein of interest

• Secondary detection: Apply biotin-conjugated secondary antibody or directly use biotin-conjugated anti-CAPN10 antibody

• Proximity labeling: The biotin moiety allows identification of proteins in close proximity to CAPN10

• Detection and analysis: Use streptavidin-conjugated enzymes or fluorophores to detect biotinylated proteins

Optimization strategies:

• Test multiple antibody concentrations to balance specific binding with background signal

• Include appropriate controls: no-primary-antibody control, isotype control, and a known interaction control

• For differential proteomics applications, consider comparing wild-type vs. CAPN10 knockdown/knockout samples to identify specific interactions

Validation approaches:

• Confirm specificity of biotinylation pattern using independent methods (co-IP, confocal microscopy)

• Analyze biotinylated proteins by mass spectrometry to identify interacting partners

• Validate key interactions using orthogonal techniques like co-immunoprecipitation or FRET

This methodology is particularly valuable for studying CAPN10 interactions with cytoskeletal components such as MAP1 family proteins and their role in actin dynamics.

How can researchers measure CAPN10 enzymatic activity using biotin-conjugated antibodies?

Measuring CAPN10 enzymatic activity presents unique challenges due to its atypical calcium-independent proteolytic mechanism. Biotin-conjugated CAPN10 antibodies can be integrated into several methodological approaches:

ELISA-based activity assays:

• Substrate capture method:

  • Immobilize known CAPN10 substrates (e.g., recombinant MAP1 family proteins) on a plate

  • Add sample containing CAPN10

  • Detect substrate cleavage using biotin-conjugated antibodies that recognize either intact substrate or cleavage products

  • Measure signal using streptavidin-HRP and appropriate substrate

• Activity-dependent antibody recognition:

  • Some antibodies may preferentially recognize the active form of CAPN10

  • Use biotin-conjugated versions of such antibodies to detect the proportion of active enzyme

In vitro digestion assay protocol:

• Prepare recombinant MAP1B (or other known CAPN10 substrate)

• Incubate with recombinant wild-type CAPN10 or inactive C73S mutant (as control)

• Test activity both with and without Ca²⁺ and with EDTA to confirm calcium independence

• Analyze cleavage products by western blot using biotin-conjugated antibodies

• Quantify the ratio of full-length to cleaved substrate as a measure of enzymatic activity

Considerations for experimental design:

• Include proper controls:

  • Wild-type CAPN10 vs. C73S mutant (substituted Cys73 by Ser at the putative catalytic center)

  • Calcium-free vs. calcium-containing conditions

  • Specific protease inhibitors

• Optimize substrate concentration and incubation time to ensure linearity of the assay

• Account for potential interactions with other calpain family members, particularly in complex samples

The C73S mutant is particularly valuable as a negative control since this mutation affects a conserved cysteine residue at the putative catalytic center of CAPN10 .

What are the applications of CAPN10 biotin-conjugated antibodies in studying actin dynamics and cytoskeletal organization?

CAPN10 biotin-conjugated antibodies provide valuable tools for studying the role of CAPN10 in regulating actin dynamics and cytoskeletal organization, particularly through its processing of MAP1 family proteins:

Immunofluorescence co-localization studies:

• Biotin-conjugated CAPN10 antibodies can be used alongside actin and tubulin markers to visualize their spatial relationships

• In wild-type cells, processed MAP1B co-localizes with tubulin, while in Capn10⁻/⁻ cells, unprocessed MAP1B co-localizes with actin stress fibers

• Protocol approach:

  • Fix cells using formaldehyde or other appropriate fixatives

  • Permeabilize cell membranes

  • Block non-specific binding sites

  • Incubate with biotin-conjugated CAPN10 antibody and other primary antibodies

  • Detect using streptavidin-conjugated fluorophores and appropriate secondary antibodies

  • Analyze using confocal microscopy

Actin dynamics studies:

• Combine biotin-conjugated CAPN10 antibodies with fluorescence recovery after photobleaching (FRAP) to assess CAPN10's role in actin reorganization

• FRAP analysis has shown that CAPN10 siRNA significantly reduces fluorescence recovery rate in cells expressing GFP-actin, indicating reduced actin dynamics

• Methodology:

  • Transfect cells with GFP-actin

  • Knockdown CAPN10 using siRNA

  • Perform FRAP on actin stress fibers

  • Measure recovery rate and mobile fraction

  • Confirm CAPN10 knockdown efficiency using biotin-conjugated antibodies

MAP1 processing analysis:

• Western blot protocol using biotin-conjugated antibodies:

  • Prepare cell lysates from control and CAPN10-manipulated samples

  • Separate proteins by SDS-PAGE

  • Transfer to membrane

  • Probe with biotin-conjugated antibodies against MAP1 family proteins

  • Detect full-length (~300 kDa) and cleaved (~34 kDa) forms

  • Quantify the ratio as an indicator of CAPN10 activity

The combination of these approaches provides comprehensive analysis of CAPN10's role in cytoskeletal dynamics.

How reliable are current CAPN10 biotin-conjugated antibodies for detecting different CAPN10 isoforms?

The reliability of CAPN10 biotin-conjugated antibodies for detecting different isoforms depends on several factors including epitope location, specificity, and validation status:

CAPN10 isoform complexity:

• Multiple alternative transcript variants have been described for the CAPN10 gene

• The CAPN10 gene contains 12 exons on chromosome 2q37.3

• Different antibodies may recognize distinct epitopes present in some but not all isoforms

Epitope considerations:

• N-terminal directed antibodies:

  • Several commercial antibodies target the N-terminal region of CAPN10

  • These may detect multiple isoforms that share the N-terminus

  • Example: Anti-CAPN10 antibody (N-Term) from St John's Laboratory (STJ91979) is generated against a synthesized peptide derived from the N-terminal region of human Calpain 10

• Full-length protein antibodies:

  • Antibodies generated against recombinant full-length protein may detect multiple epitopes

  • Example: CUSABIO's biotin-conjugated CAPN10 antibody (CSB-PA864027LD01HU) uses recombinant Human Calpain-10 protein (1-287AA) as immunogen

Molecular weight detection:

• Calculated molecular weight of CAPN10 is approximately 75 kDa

• Observed molecular weights vary:

  • Some antibodies detect CAPN10 at approximately 57 kDa

  • Other observed weights range from 39-75 kDa depending on the isoform and detection method

Validation status:

• Check manufacturer validation data for each antibody

• Western blot validation in multiple cell lines helps confirm specificity

• Cross-validation with antibodies targeting different epitopes

• Validation in knockout/knockdown samples is particularly valuable:

  • CAPN10 siRNA treatment should reduce or eliminate the target band

  • Samples from Capn10⁻/⁻ mice serve as excellent negative controls

When selecting a biotin-conjugated CAPN10 antibody for isoform detection, researchers should carefully review the immunogen information and validation data to ensure the antibody can detect their isoform(s) of interest.

What are the key technical considerations for performing co-immunoprecipitation studies with biotin-conjugated CAPN10 antibodies?

Co-immunoprecipitation (Co-IP) using biotin-conjugated CAPN10 antibodies requires careful optimization to identify interacting partners while minimizing background. Here are the key technical considerations:

Protein complex preservation:

• Lysis buffer selection:

  • Use gentle, non-denaturing lysis buffers that preserve protein-protein interactions

  • Consider buffers containing 1% NP-40 or 0.5% Triton X-100 with physiological salt concentrations

  • Include protease inhibitors to prevent degradation of CAPN10 and its interacting partners

• Calcium considerations:

  • While CAPN10 is atypical and can function without calcium, include calcium chelators (EDTA/EGTA) in some conditions to distinguish calcium-dependent and independent interactions

  • Compare results with and without calcium to identify differential interactions

Pull-down strategy optimization:

• Direct vs. indirect approach:

  • Direct: Use biotin-conjugated CAPN10 antibody for direct capture followed by streptavidin beads

  • Indirect: Use unconjugated CAPN10 antibody for capture, followed by biotin-conjugated secondary antibody and streptavidin beads

• Controls:

  • IgG control: Use species-matched biotin-conjugated IgG

  • Blocking peptide control: Pre-incubate antibody with immunizing peptide

  • CAPN10 knockout/knockdown samples: Ideal negative control

  • Input sample: Include to verify protein expression

Elution and detection strategies:

• Gentle elution methods:

  • Competition with biotin: For streptavidin-biotin based systems

  • Mild elution buffers: Maintain integrity of co-immunoprecipitated complexes

• Western blot detection:

  • Avoid detecting the heavy and light chains of the IP antibody by using:

    • HRP-conjugated protein A/G

    • Light chain-specific secondary antibodies

    • TrueBlot® detection system

• Mass spectrometry preparation:

  • Consider on-bead digestion to minimize contamination

  • Include crosslinking before lysis for transient interactions

Validation of interactions:

• Perform reciprocal Co-IP experiments (pull down with antibody against interacting protein)

• Confirm interactions with known partners like MAP1 family proteins

• Use proximity ligation assay (PLA) as an orthogonal method to validate interactions in situ

When specifically investigating interactions between CAPN10 and MAP1 family proteins, researchers should include controls to distinguish between interactions with full-length versus processed forms of these proteins.

How can researchers troubleshoot issues with biotin-conjugated CAPN10 antibodies in western blot applications?

When troubleshooting issues with biotin-conjugated CAPN10 antibodies in western blot applications, consider the following methodological approaches to common problems:

High background signal:

• Streptavidin reactivity optimization:

  • Increase blocking time/concentration (use 5% BSA in TBS-T)

  • Add avidin/biotin blocking step to reduce endogenous biotin

  • Try different streptavidin-HRP dilutions (typically 1:5000-1:20000)

  • Consider adding 0.05-0.1% SDS to the antibody diluent

• Washing optimization:

  • Increase number and duration of washes

  • Use higher salt concentration in wash buffer

  • Add 0.05% SDS to wash buffer for more stringent washing

Weak or no signal:

• Sample preparation:

  • Verify protein expression in your sample (use positive control)

  • Optimize protein loading (30-50 μg total protein typically)

  • Consider enrichment methods before loading

  • Ensure complete protein transfer (stain membrane post-transfer)

• Antibody detection system:

  • Try more sensitive ECL reagents

  • Consider using streptavidin-poly-HRP for signal amplification

  • Optimize antibody concentration (try 1:500-1:2000 dilution range)

  • Extend primary antibody incubation (overnight at 4°C)

Wrong molecular weight:

• CAPN10 calculated molecular weight is approximately 75 kDa

• Observed molecular weights may vary:

  • Some antibodies detect CAPN10 at approximately 57 kDa

  • Check literature for expected band sizes in your specific cell type

  • Consider multiple isoforms or post-translational modifications

  • Verify with other validated CAPN10 antibodies

Multiple bands:

• Distinguish between isoforms and degradation products by:

  • Including freshly prepared lysates with protease inhibitors

  • Comparing with recombinant protein control

  • Performing peptide competition assay

  • Testing in knockout/knockdown samples

Protocol optimization checklist:

• Sample preparation: Fresh preparation, include protease inhibitors

• Protein loading: 30-50 μg total protein per lane

• Transfer conditions: Semi-dry or wet transfer optimized for high molecular weight proteins

• Blocking: 5% BSA in TBS-T for 1-2 hours at room temperature

• Primary antibody: 1:500-1:2000 dilution, overnight at 4°C

• Washing: 3-5 times, 5-10 minutes each with TBS-T

• Detection reagent: Streptavidin-HRP at 1:5000-1:20000

• Exposure: Start with short exposures and increase as needed