CAPN10 Antibody, FITC conjugated

What is CAPN10 and why is it significant for diabetes research?

CAPN10 was identified as the first candidate susceptibility gene for type 2 diabetes mellitus (T2DM) through positional cloning. The molecular mechanisms linking CAPN10 to T2DM involve its role in processing microtubule-associated protein 1 (MAP1) family proteins, which affects cytoskeletal dynamics and insulin secretion . CAPN10 regulates actin dynamics via MAP1B cleavage, and deficiency in calpain-10 expression may affect insulin secretion by abnormal actin reorganization, coordination, and dynamics through MAP1 family processing .

Key research findings show:

• CAPN10 knockout mice exhibit significantly increased insulin secretion at both high and low glucose levels

• In Capn10−/− mouse embryonic fibroblasts, MAP1B is abnormally localized at actin filaments rather than at microtubules

• CAPN10 is a calcium-regulated non-lysosomal thiol-protease catalyzing limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction

How do FITC-conjugated CAPN10 antibodies differ from other detection methods?

FITC-conjugated CAPN10 antibodies offer direct visualization without requiring secondary antibodies, which provides several methodological advantages:

For optimal results when using FITC-conjugated antibodies:

• Store in light-protected vials or covered with aluminum foil

• Stable for at least 12 months at 4°C

• For longer storage (24 months), dilute with up to 50% glycerol and store at -20°C to -80°C

• Note that freezing and thawing conjugated antibodies may compromise enzyme activity and antibody binding

How should multicolor flow cytometry experiments using CAPN10-FITC antibodies be designed?

When designing multicolor flow cytometry experiments incorporating CAPN10-FITC antibodies, proper controls are essential for accurate interpretation:

Recommended control strategy:

• Include Fluorescence Minus One (FMO) controls for each fluorochrome in your panel:

  • For a 4-color panel (e.g., CAPN10-FITC, CD4-PerCP, CD8-Pacific Blue, CD25-PE), prepare the following tubes :

    • Tube 1: All antibodies except FITC

    • Tube 2: All antibodies except PerCP

    • Tube 3: All antibodies except Pacific Blue

    • Tube 4: All antibodies except PE

    • Tube 5: Complete panel

• For activation markers, additional controls are necessary:

  • Include Fc receptor blocking before staining

  • Prepare tubes with blocking antibody (no fluorescent conjugate) to control for non-specific binding

  • Consider using matched isotype controls with the same F/P ratio

• Spectral considerations:

  • FITC excites at 488nm and emits at approximately 525nm

  • Ensure minimal spectral overlap with other fluorophores in your panel

  • Apply proper compensation controls using single-stained samples

What are the optimal fixation and permeabilization conditions for CAPN10-FITC immunofluorescence studies?

CAPN10 is primarily expressed in liver, skeletal muscle, and pancreatic islets and functions as a calcium-regulated intracellular signaling protease . For successful immunofluorescence studies:

Methodological approach:

• Fixation options:

  • 4% paraformaldehyde (10-15 minutes at room temperature) preserves cell morphology while maintaining antibody epitope accessibility

  • Methanol fixation (100% methanol, -20°C, 10 minutes) may enhance nuclear and cytoskeletal protein detection

• Permeabilization considerations:

  • Since CAPN10 is an intracellular protease, permeabilization is essential

  • 0.1-0.5% Triton X-100 (5-10 minutes) provides adequate permeabilization for cytoplasmic proteins

  • 0.05-0.1% saponin maintains better morphology but requires presence in all buffers

• Buffer composition:

  • Use PBS with 0.02% sodium azide and include 50% glycerol for storage solutions

  • Blocking with 1-5% BSA reduces non-specific binding

• Controls:

  • Include CAPN10 knockout or knockdown samples as negative controls

  • Implement peptide competition controls to validate antibody specificity

How can researchers differentiate between CAPN10 splice variants in their experiments?

CAPN10 has multiple splice variants, which can complicate interpretation of experimental results. A methodological approach to differentiate these variants includes:

• Molecular weight analysis:

  • Full-length CAPN10 has a calculated molecular weight of 75 kDa

  • Observed molecular weight can differ depending on post-translational modifications

  • Western blot analysis typically shows bands at approximately 57-68 kDa

• Epitope mapping:

  • Verify which region of CAPN10 your antibody targets (N-terminal, C-terminal, etc.)

  • Some antibodies target the N-terminal region (amino acids 1-287) while others target specific sequences like "RRPQEICATPRLFPDDPREGQVKQGLLGDCWFLCACAALQKSRHLLDQVI"

• RT-PCR analysis:

  • Design primers that span exon-exon junctions specific to different splice variants

  • Validate findings with sequencing of PCR products

• Cross-validation:

  • Use multiple antibodies targeting different epitopes to confirm findings

  • Compare results from protein (Western blot) and mRNA (RT-PCR) analyses

How do you interpret contradictory results between CAPN10 genotype and phenotype in different populations?

Research has shown variable associations between CAPN10 polymorphisms and type 2 diabetes across different populations, requiring careful interpretation:

Methodological approach to resolving contradictions:

• Population-specific haplotype analysis:

  • The 112/121 diplotype of CAPN10 is associated with a 3-fold increase in T2DM risk in Mexican-Americans and Northern Europeans

  • A novel 111/121 diplotype is associated with T2DM in the Korean population

  • Allele frequencies of UCSNP-44, -43, -19, and -63 polymorphisms vary significantly across populations (e.g., G allele frequency of UCSNP-43 ranges from 0.62 in Pima Indians to 0.96 in Japanese)

• Functional validation studies:

  • Assess CAPN10 expression levels in different tissues

  • Measure enzymatic activity of CAPN10 variants

  • Evaluate substrate processing efficiency (e.g., MAP1 family proteins)

• Interaction analysis:

  • Consider gene-gene interactions

  • Evaluate gene-environment interactions

  • Assess the impact of different genetic backgrounds

• Methodological reconciliation strategies:

  • Meta-analysis of existing studies

  • Stratification by ethnicity, age, BMI, and other relevant factors

  • Adjustment for multiple hypothesis testing

How does calcium dependency affect CAPN10 activity in experimental systems?

Unlike typical calpain family members, CAPN10 exhibits unique calcium requirements that impact experimental design:

Methodological implications:

• In vitro proteolytic activity:

  • CAPN10 can cleave MAP1B both with and without Ca²⁺

  • CAPN10 maintains proteolytic activity even in the presence of 5 mM EDTA

  • This is distinct from typical calpain members that require calcium for activation

• Structural considerations:

  • CAPN10 lacks the penta-EF-hand calcium-binding motif found in typical calpain members

  • This structural difference explains its calcium-independent proteolytic activity

• Experimental design adjustments:

  • Buffer composition should be carefully considered

  • Control experiments should include both calcium-containing and calcium-free conditions

  • When studying CAPN10 alongside other calpains, consider differential calcium requirements

• Interaction with inhibitors:

  • Short-term exposure to calpain inhibitors enhances insulin secretion through accelerated exocytosis

  • Long-term (48h) exposure suppresses glucose-stimulated insulin secretion

What are the best approaches for visualizing CAPN10-mediated actin dynamics using FITC-conjugated antibodies?

CAPN10 regulates actin dynamics through MAP1B processing, and visualization of these dynamics requires specialized approaches:

Methodological recommendations:

• Live cell imaging techniques:

  • Combine CAPN10-FITC antibodies with live-cell actin markers like SiR-Actin

  • Implement FRAP (Fluorescence Recovery After Photobleaching) analysis to measure actin dynamics

  • Knockdown studies have shown that CAPN10 siRNA treatment significantly reduces fluorescence recovery rate in FRAP experiments

• Co-localization studies:

  • In wild-type cells, processed MAP1B co-localizes with tubulin

  • In Capn10⁻/⁻ MEF cells, unprocessed MAP1B co-localizes with actin stress fibers

  • Use high-resolution microscopy (confocal, STED, or SIM) for accurate co-localization analysis

• Experimental validation approaches:

  • Compare actin dynamics in control vs. CAPN10 knockdown/knockout models

  • Rescue experiments with wild-type vs. mutant CAPN10 (e.g., C73S catalytic mutant)

  • Utilize MAP1B mutants resistant to CAPN10 cleavage (e.g., M2219P mutation)

• Quantitative analysis methods:

  • Measure stress fiber thickness and distribution

  • Calculate fluorescence recovery half-time and mobile fraction in FRAP experiments

  • Quantify co-localization using Pearson's or Manders' coefficients

How can CAPN10-FITC antibodies be utilized to investigate the molecular mechanisms linking CAPN10 to insulin secretion?

CAPN10's role in insulin secretion can be studied using FITC-conjugated antibodies through multiple experimental approaches:

Methodological framework:

• Pancreatic islet immunofluorescence:

  • Co-stain pancreatic islets with CAPN10-FITC and insulin antibodies

  • Compare wild-type vs. diabetic models to assess CAPN10 expression and localization

  • Quantify co-localization in different glucose conditions

• Actin cytoskeleton dynamics in β-cells:

  • Previous studies have shown that inhibition of calpain activities impairs actin reorganization and insulin secretion from β-cells

  • Visualize actin reorganization during glucose-stimulated insulin secretion

  • Compare cytoskeletal changes in models with varying CAPN10 expression levels

• Analysis of MAP1 processing:

  • Examine the relationship between MAP1 processing status and insulin secretion

  • Western blot analysis can detect processing of MAP1 family proteins into heavy and light chains

  • Correlate processing efficiency with insulin secretion measurements

• Insulin granule trafficking:

  • Track insulin granule movement in relation to CAPN10-mediated cytoskeletal changes

  • Enhancement of insulin secretion by short-term exposure to calpain inhibitors is mediated by accelerated exocytosis of insulin granules

What are the most effective controls for validating CAPN10 antibody specificity in diabetes research?

Ensuring antibody specificity is critical for reliable research outcomes, particularly in diabetes studies:

Validation methodological approaches:

• Genetic controls:

  • Use Capn10⁻/⁻ mouse embryonic fibroblasts as negative controls

  • Implement CAPN10 siRNA knockdown experiments with multiple independent siRNAs

  • Compare results with heterozygous models to assess dose-dependent effects

• Molecular validation:

  • Express recombinant CAPN10 with known tags for independent verification

  • Utilize the C73S mutant (substituted Cys73 by Ser), which lacks proteolytic activity

  • Perform peptide competition assays using the immunizing peptide

• Cross-reactivity assessment:

  • Test antibody against related calpain family members

  • While there are 15 calpain members, research shows that none except CAPN3 can cleave MAP1B

  • Verify species cross-reactivity through sequence alignment (antibodies may show different reactivity levels with human, mouse, rat, etc.)

• Functional validation:

  • Confirm that the antibody detects functional differences in CAPN10 activity

  • Verify that the FITC conjugation doesn't interfere with epitope binding

  • Compare results with unconjugated antibodies to ensure conjugation doesn't affect specificity