TNNI3 Antibody, FITC conjugated

What is TNNI3 and why is it important in cardiac research?

TNNI3 (troponin I type 3) is the gene encoding cardiac troponin I (cTnI), a pivotal component of the sarcomeric structure in the myocardium. The full-length protein is 210 amino acids in length with a molecular mass of approximately 24 kDa . Unlike skeletal muscle troponin I isoforms, cardiac troponin I contains a unique N-terminal extension that serves as an important regulatory structure containing protein kinase A (PKA) target serine residues . This cardiac-specific isoform is exclusively expressed in heart tissue and plays a critical role in cardiac muscle contraction regulation.

The significance of TNNI3 in cardiac research stems from:

• Its role as a cardiac-specific biomarker

• Its involvement in calcium-mediated regulation of muscle contraction

• The association between TNNI3 mutations and various cardiomyopathies including hypertrophic, restrictive, and dilated forms

What are the key specifications of FITC-conjugated TNNI3 antibodies?

FITC-conjugated TNNI3 antibodies typically have the following specifications:

Source: Product specifications from antibody suppliers

What applications are FITC-conjugated TNNI3 antibodies validated for?

FITC-conjugated TNNI3 antibodies are validated for multiple applications, with particular utility in applications requiring direct visualization without secondary antibodies:

• Immunofluorescence microscopy

• Flow cytometry

• Immunohistochemistry (fluorescent)

• Confocal microscopy of cardiac tissue sections

• Direct visualization of cardiomyocytes in differentiation studies from stem cells

Unlike unconjugated antibodies that require secondary detection, FITC-conjugated antibodies provide direct visualization, simplifying protocols and reducing non-specific background in multi-color staining experiments .

How should I optimize immunofluorescence protocols when using TNNI3-FITC antibodies for cardiac tissue?

Optimizing immunofluorescence protocols for TNNI3-FITC antibodies in cardiac tissue requires careful consideration of several parameters:

Tissue Preparation:

• Use fresh or properly fixed tissue (4% paraformaldehyde is commonly used)

• For paraffin-embedded sections, perform heat-mediated antigen retrieval using citrate buffer (pH 6.0) or TE buffer (pH 9.0)

• Section thickness of 5-7 μm is optimal for antibody penetration and imaging

Staining Protocol Optimization:

• Block with 5-10% normal serum from a species different from the host antibody species

• Start with manufacturer's recommended dilution (typically 1:300-1:1200 for IF) and adjust based on signal-to-noise ratio

• Incubate at 4°C overnight or room temperature for 2-3 hours

• Include a nuclear counterstain (DAPI) for visualization of tissue architecture

• Mount using an anti-fade mounting medium specifically formulated for fluorescent preservation

Controls to Include:

• Positive control: Known TNNI3-expressing tissue (human or mouse heart tissue)

• Negative control: Tissue known not to express TNNI3

• Secondary-only control: Omitting primary antibody to assess background

• Isotype control: Using irrelevant FITC-conjugated antibody of same isotype

When visualizing cardiomyocytes differentiated from iPSCs, co-staining with cardiac troponin T can help confirm cardiomyocyte identity, as demonstrated in validation studies .

What are the considerations for using TNNI3-FITC antibodies in multi-color immunofluorescence experiments?

When designing multi-color immunofluorescence experiments incorporating TNNI3-FITC antibodies, consider:

Spectral Compatibility:

• FITC emission (515 nm) overlaps partially with other green fluorophores

• Avoid fluorophores with emissions between 510-550 nm on other antibodies

• Compatible partners include red fluorophores (e.g., NorthernLights 557) and far-red dyes (e.g., Alexa Fluor 647)

Sequential Staining Strategy:

• For multiple mouse-derived antibodies, use a sequential staining approach

• Apply FITC-conjugated TNNI3 antibody first

• Block with excess anti-mouse IgG before applying other mouse antibodies

• Use directly conjugated antibodies for other targets when possible to avoid cross-reactivity

Panel Design Example:

As demonstrated in R&D Systems validation data, this approach allows clear visualization of striated pattern characteristic of cardiac troponin localization in cardiomyocytes .

How can I quantify TNNI3 expression levels using FITC-conjugated antibodies?

Quantification of TNNI3 expression using FITC-conjugated antibodies can be performed through several methods:

Flow Cytometry Quantification:

• Dissociate cardiac tissue or cultured cardiomyocytes into single-cell suspension

• Fix and permeabilize cells (recommended: 4% paraformaldehyde followed by 0.1% Triton X-100)

• Incubate with TNNI3-FITC antibody at optimized concentration

• Include unstained and isotype controls

• Measure mean fluorescence intensity (MFI) and percent positive cells

• Use standardized beads to convert MFI to molecules of equivalent soluble fluorochrome (MESF)

Fluorescence Microscopy Quantification:

• Capture images using consistent exposure settings across all samples

• Analyze using ImageJ or similar software:

  • Define regions of interest (ROIs) around cardiomyocytes

  • Measure integrated density and area

  • Subtract background fluorescence

  • Calculate corrected total cell fluorescence (CTCF) using the formula:
    CTCF = Integrated Density - (Area × Mean background fluorescence)

• Compare CTCF values across experimental conditions

Considerations for Accurate Quantification:

• Include calibration standards in each experiment

• Account for photobleaching by minimizing exposure time

• Ensure consistent antibody concentration and incubation times

• Use appropriate statistical analysis for comparing conditions

How can TNNI3-FITC antibodies be used to study cardiomyopathy disease mechanisms?

TNNI3-FITC antibodies provide valuable tools for investigating cardiomyopathy disease mechanisms, particularly those caused by TNNI3 mutations:

Engineered Heart Tissue (EHT) Models:
Recent research has utilized TNNI3 antibodies to visualize protein localization in EHT models created from patient-derived iPSCs carrying TNNI3 mutations . These models recapitulate hallmarks of restrictive cardiomyopathy, including:

• Impaired relaxation in cardiac tissue

• Altered calcium handling

• Sarcomeric disorganization

Mutation-Specific Studies:
TNNI3-FITC antibodies can be used to examine how specific mutations (e.g., R170W, R186Q) affect:

• Protein localization within the sarcomere

• Co-localization with binding partners

• Stability and turnover rates of the protein

• Response to pharmaceutical interventions

For example, studies have shown that the TNNI3 p.R186Q mutation disrupts EGFR and cTnI interaction, leading to abnormal fatty acid metabolism in cardiomyocytes—a finding visualized using fluorescently-labeled antibodies .

Gene Correction Studies:
FITC-labeled TNNI3 antibodies have been instrumental in demonstrating that:

• Gene correction of TNNI3 mutations improves relaxation impairment in iPSC-derived cardiomyocytes

• Overexpression of wild-type TNNI3 can ameliorate the phenotype associated with dominant negative mutations

• Expression levels of corrected protein correlate with functional improvement

What are the considerations when using TNNI3-FITC antibodies to assess cardiomyocyte maturation?

TNNI3 expression serves as an important marker of cardiomyocyte maturation. During development, there is an isoform switch from TNNI1 (fetal/slow skeletal) to TNNI3 (cardiac), making TNNI3-FITC antibodies valuable for assessing maturation status :

Maturation Assessment Protocol:

• Co-stain iPSC-derived cardiomyocytes with antibodies against both TNNI1 and TNNI3

• Quantify expression ratio over time during differentiation

• Correlate with functional parameters (calcium handling, contractility)

• Compare expression patterns with primary cardiac tissue controls

Key Considerations:

• Expression level is time-dependent (increases with maturation)

• Subcellular localization becomes more organized (striated pattern develops)

• Sarcomeric organization correlates with functional maturity

• Post-translational modifications (phosphorylation states) change during maturation

• Combined with other markers (e.g., MYH6/MYH7 ratio) provides comprehensive maturation assessment

Sample Analysis:

Researchers should note that current in vitro differentiation protocols may not achieve complete maturation equivalent to adult cardiac tissue .

How can I use TNNI3-FITC antibodies to investigate post-translational modifications of cardiac troponin I?

Post-translational modifications (PTMs) of cardiac troponin I significantly impact cardiac function. TNNI3-FITC antibodies can be used alongside modification-specific antibodies to investigate these critical regulatory mechanisms:

Dual Labeling Strategy:

• Use TNNI3-FITC antibody to localize total cardiac troponin I

• Co-stain with non-FITC conjugated PTM-specific antibodies (e.g., phospho-serine 23/24 antibodies)

• Assess co-localization and relative abundance of modified versus unmodified protein

PTMs of Interest in cTnI Research:

• Phosphorylation at Ser23/24 (PKA-mediated, β-adrenergic response)

• Phosphorylation at Ser43/45 and Thr144 (PKC-mediated)

• O-GlcNAcylation at Ser/Thr residues

• Proteolytic cleavage (C-terminal degradation)

Advanced Application - FRET Analysis:
For studying protein-protein interactions involving TNNI3:

• Use TNNI3-FITC as donor fluorophore

• Label interaction partner with acceptor fluorophore (e.g., Cy3, Rhodamine)

• Measure fluorescence resonance energy transfer (FRET) efficiency

• Calculate molecular proximity based on FRET efficiency

Research has demonstrated that phosphorylation status of TNNI3 at specific residues directly impacts calcium sensitivity and relaxation kinetics in cardiomyocytes .

How can I address weak or absent signals when using TNNI3-FITC antibodies?

When experiencing weak or absent signals with TNNI3-FITC antibodies, consider these systematic troubleshooting steps:

Sample Preparation Issues:

• Inadequate fixation: Ensure proper fixation with 4% paraformaldehyde for 15-20 minutes

• Insufficient permeabilization: Optimize detergent concentration (0.1-0.5% Triton X-100) and duration

• Overfixation: Excessive fixation can mask epitopes; reduce fixation time or perform antigen retrieval

• Improper antigen retrieval: For FFPE tissues, use heat-mediated retrieval with citrate buffer (pH 6.0) or TE buffer (pH 9.0)

Antibody-Related Issues:

• Antibody degradation: Check for exposure to light or improper storage conditions

• Insufficient concentration: Titrate antibody using a range of concentrations (1:100 to 1:1000)

• Epitope accessibility: Try alternative antibody clones recognizing different epitopes

• FITC photobleaching: Minimize exposure to light during procedures and use anti-fade mounting media

Protocol Modifications to Improve Signal:

• Increase antibody incubation time (overnight at 4°C)

• Use signal amplification systems (e.g., tyramide signal amplification)

• Optimize blocking conditions (5-10% normal serum from species different from host)

• Try different fixation protocols (methanol vs. paraformaldehyde)

• Adjust imaging settings (longer exposure, higher gain) within linear range

Control Experiments:

• Perform parallel staining with unconjugated TNNI3 antibody and fluorescent secondary

• Test antibody on known positive control (heart tissue sections)

• Confirm target protein expression by Western blot

How do I differentiate between specific and non-specific signals when using TNNI3-FITC antibodies?

Differentiating specific from non-specific signals is critical for accurate interpretation of results:

Controls to Implement:

• Isotype control: Use FITC-conjugated antibody of same isotype but irrelevant specificity

• Absorption control: Pre-incubate antibody with excess recombinant TNNI3 protein

• Knockout/knockdown control: Use tissue or cells lacking TNNI3 expression

• Secondary-only control: Omit primary antibody to assess background fluorescence

• Competitive binding assay: Co-incubate with unlabeled TNNI3 antibody

Characteristics of Specific Staining:

• Localization consistent with known biology (sarcomeric striation pattern)

• Signal correlates with expected expression level across tissues

• Co-localization with other cardiac markers (e.g., cardiac troponin T)

• Signal absent in negative control tissues

• Consistent results across multiple antibody clones

Addressing Common Sources of Non-Specific Signal:

• Autofluorescence: Include unstained control and consider autofluorescence quenching treatments

• Fc receptor binding: Include Fc receptor blocking step before antibody incubation

• Hydrophobic interactions: Increase detergent concentration in wash buffers

• Dead cell binding: Remove dead cells before staining or use viability dyes

What are the best practices for quantitative comparison of TNNI3 expression across different experimental conditions?

For reliable quantitative comparison of TNNI3 expression across experimental conditions:

Standardization Practices:

• Process all samples simultaneously using identical protocols

• Include calibration standards in each experiment

• Maintain consistent antibody lots and concentrations

• Use internal controls (housekeeping proteins) for normalization

• Analyze images blinded to experimental conditions

Image Acquisition Guidelines:

• Use identical microscope settings for all samples:

  • Exposure time

  • Gain

  • Offset

  • Laser power (for confocal)

• Capture multiple representative fields per sample (minimum 5-10)

• Include reference samples in each imaging session

• Monitor for photobleaching during acquisition

Quantification Protocol:

• Use automated analysis software to reduce bias

• Define consistent thresholding parameters across all images

• Analyze signal intensity and distribution patterns:

  • Mean fluorescence intensity

  • Integrated density

  • Area of positive staining

  • Pattern recognition (striated vs. diffuse)

• Apply appropriate statistical analysis (ANOVA with post-hoc tests for multiple comparisons)

Reporting Standards:

• Document all methods in detail, including antibody information (clone, lot, concentration)

• Report all normalization procedures

• Include both representative images and quantitative data

• Present results with appropriate statistical analysis

How are TNNI3-FITC antibodies being used in engineered heart tissue (EHT) models of cardiomyopathy?

Recent advances in engineered heart tissue (EHT) models have leveraged TNNI3-FITC antibodies to investigate cardiac pathophysiology:

Current Applications in EHT Research:

• Visualizing sarcomeric organization in 3D cardiac tissues

• Tracking TNNI3 localization in disease-specific iPSC-derived cardiomyocytes

• Monitoring effects of genetic corrections on protein expression and localization

• Assessing drug responses at the protein level

A recent groundbreaking study demonstrated that EHT can precisely recapitulate the impaired relaxation phenotype of restrictive cardiomyopathy (RCM) in vitro using tissues generated from patient-derived iPSCs carrying TNNI3 mutations . The researchers used fluorescently-labeled antibodies to:

• Confirm successful genetic correction of TNNI3 mutations

• Visualize improvements in sarcomeric organization

• Correlate protein expression with functional recovery

• Demonstrate rescue of phenotype through wild-type TNNI3 overexpression

This approach has opened new avenues for personalized medicine approaches to cardiac disease modeling and therapeutic development.

What advances have been made in understanding TNNI3 mutations and their relationship to cardiomyopathies?

Recent research using TNNI3 antibodies has significantly advanced our understanding of TNNI3 mutation pathophysiology:

Key Recent Findings:

• Biallelic TNNI3 null mutations cause severe forms of neonatal dilated cardiomyopathy

• The TNNI3 p.R186Q mutation promotes hypertrophic cardiomyopathy through abnormal fatty acid metabolism

• Point mutations in TNNI3 can disrupt protein-protein interactions with key regulatory partners

• Some mutations show differential effects based on heterozygous vs. homozygous expression

Molecular Mechanisms Elucidated:

• The p.R186Q mutation disrupts EGFR-cTnI binding, leading to increased FASN expression and abnormal lipid metabolism in cardiomyocytes

• Homozygous null mutations (p.Arg98*, p.Arg69Alafs*8) cause early-onset dilated cardiomyopathy through loss of functional protein

• Some mutations demonstrate low penetrance in heterozygous carriers but severe phenotypes in homozygotes

These findings highlight the complexity of TNNI3-related cardiomyopathies and demonstrate that different mutations may operate through distinct pathological mechanisms.

How might TNNI3-FITC antibodies contribute to developing therapeutics for TNNI3-associated cardiomyopathies?

TNNI3-FITC antibodies are playing crucial roles in developing potential therapeutics for TNNI3-associated cardiomyopathies:

Current Therapeutic Development Approaches:

• Gene Therapy Screening: TNNI3-FITC antibodies enable visualization of protein expression following gene therapy approaches (viral vectors delivering wild-type TNNI3)

• Small Molecule Drug Screening: Fluorescent antibodies allow high-content screening to identify compounds that correct protein mislocalization or improve sarcomere organization

• Gene Editing Validation: FITC-labeled antibodies confirm successful CRISPR-mediated gene correction through proper protein localization and expression levels

• Antisense Oligonucleotide Development: Antibodies help visualize allele-specific silencing of mutant TNNI3 while preserving wild-type expression

Proof-of-Concept Findings:
Recent research demonstrated that overexpression of wild-type TNNI3 improved relaxation impairment in engineered heart tissues carrying the R170W mutation . This effect was visualized and quantified using fluorescently-labeled antibodies that showed:

• Increased TNNI3 expression levels

• Improved sarcomeric organization

• Normalized calcium handling

• Rescued mechanical function

These findings establish the foundation for therapeutic approaches focused on increasing wild-type TNNI3 expression to overcome dominant-negative effects of certain mutations.

How has TNNI3 evolved across species and what implications does this have for research using different model organisms?

Recent evolutionary studies of TNNI3 have revealed important insights for researchers using model organisms:

Evolutionary Conservation:
The TNNI3 gene belongs to a complex gene family with a distinct evolutionary history. Recent phylogenetic analyses across vertebrates have revealed:

• Five distinct TNNI classes (TNNI1-5) exist across vertebrates

• TNNI3 encoding "cardiac TnI" in tetrapods was independently lost in cartilaginous and ray-finned fishes

• Ray-finned fishes predominantly express TNNI1 in the heart instead of TNNI3

• Sharks express TNNI5 in hearts, which contains an N-terminal extension similar to tetrapod TNNI3

Implications for Antibody Selection:
These evolutionary differences have important implications for researchers:

• Antibodies raised against mammalian TNNI3 may not cross-react with fish cardiac troponin I

• Different model organisms may express different troponin I isoforms in the heart

• The regulatory mechanisms may differ across vertebrate lineages

• Developmental timing of isoform switching varies across species

Cross-Reactivity Table for Common Research Models:

Based on these considerations, researchers should carefully validate antibodies when working with non-mammalian models .

What are the unique features of the TNNI3 N-terminal extension and their functional significance?

The N-terminal extension of TNNI3 has long been considered unique to mammalian cardiac troponin I, but recent evolutionary analysis has revealed new insights:

Structure and Conservation:

• Previously thought to be unique to TNNI3, N-terminal extensions have now been discovered in TNNI5 proteins in sharks

• This suggests the N-terminal extension may be an ancestral feature rather than a mammalian innovation

• The extension contains key phosphorylation sites (Ser23/24 in human TNNI3)

• The region is approximately 30 amino acids in length and extends beyond the core structure

Functional Significance:
The N-terminal extension serves critical regulatory functions:

• Contains PKA phosphorylation sites activated during β-adrenergic stimulation

• Phosphorylation decreases myofilament Ca²⁺ sensitivity

• This decreased sensitivity increases rate of relaxation during diastole

• Contributes to the "fight-or-flight" cardiac response

• Mediates protein-protein interactions with other cardiac regulatory proteins

Research Implications:
Understanding the evolutionary context of the N-terminal extension:

• Challenges previous assumptions about cardiac regulation in non-mammalian vertebrates

• Suggests potential for comparative studies of cardiac regulation across diverse vertebrates

• Indicates that different mechanisms may have evolved for adrenergic regulation in fish hearts

• Provides context for interpretation of results when using antibodies targeting this region

When using TNNI3-FITC antibodies targeting epitopes in the N-terminal region, researchers should consider these evolutionary differences in experimental design and interpretation .