OBSCN Antibody, FITC conjugated

What is OBSCN and why is it significant in scientific research?

OBSCN (Obscurin) is a giant protein (ranging from ~70-870 kDa) encoded by the single OBSCN gene spanning 150 kb on human chromosome 1q42. It undergoes complex alternative splicing, giving rise to at least 4 isoforms . Obscurin-A (~720 kDa) has a modular architecture with tandem adhesion and signaling motifs. The NH2-terminal half contains repetitive immunoglobulin (Ig) and fibronectin-III (FN-III) domains, while its COOH-terminal portion consists of signaling domains, including calmodulin-binding domains, src-homology-3 domains, and tandem Rho-guanine nucleotide exchange factor (Rho-GEF) and pleckstrin homology motifs . Obscurin is significant in research as it exhibits close association with large proteins and interacts with complexes of titin, myomesin, and small ankyrin-1 in striated muscles, serving as a structural protein connecting the sarcomere M-line with the sarcoplasmic reticulum .

What is the principle behind FITC conjugation and how does it enable detection of OBSCN?

FITC (fluorescein isothiocyanate) is a fluorochrome dye that enables visualization of antibody binding through fluorescence microscopy. The conjugation occurs through free amino groups of proteins or peptides . FITC absorbs ultraviolet or blue light, exciting molecules which then emit visible yellow-green light at peak wavelengths of approximately 495nm (excitation) and 525nm (emission) . When the excitation light is removed, the emission light ceases. This property allows researchers to visualize OBSCN protein localization in cells and tissues with high sensitivity. The FITC conjugation process to proteins is relatively simple and usually does not alter the biological activity of the labeled protein .

How should proper controls be established when using OBSCN antibody, FITC conjugated?

When designing experiments with OBSCN antibody, FITC conjugated, multiple controls should be implemented:

• Negative controls: Include samples without primary antibody but with all other reagents to assess background fluorescence.

• Isotype controls: Use an irrelevant FITC-conjugated antibody of the same isotype (e.g., IgG for OBSCN antibody) to confirm specificity of binding, as demonstrated in flow cytometry experiments where cells labeled with isotype control and stained with FITC-conjugated secondary antibody produce a distinct peak pattern from specific antibody binding .

• Blocking peptide controls: Pre-incubate the antibody with the immunizing peptide to confirm binding specificity.

• Known positive and negative tissue/cell controls: Include samples with confirmed high and low/absent OBSCN expression.

• Fluorescence quenching control: As demonstrated in flow cytometry experiments, pre-incubation with anti-FITC antibody can quench fluorescence signals, providing validation of specific FITC-conjugated antibody binding .

What are the optimal storage conditions for maintaining FITC-conjugated antibody activity?

To maintain optimal activity of OBSCN antibody, FITC conjugated:

• Store at 4°C in the dark to prevent photobleaching, as continuous exposure to light causes the FITC-conjugated antibody to gradually lose its fluorescence .

• For long-term storage, keep at -20°C or -80°C, and avoid repeated freeze-thaw cycles .

• The antibody is typically supplied in a buffer containing preservatives like 0.03% Proclin 300 and stabilizers such as 50% Glycerol in PBS at pH 7.4 .

• When using antibodies containing sodium azide as a preservative (0.01%), be aware that azide is incompatible with FITC conjugation reactions and should be removed by dialysis against PBS before any further conjugation procedures .

What protocol should be followed for immunofluorescence using OBSCN antibody, FITC conjugated?

A standardized protocol for immunofluorescence using OBSCN antibody, FITC conjugated:

• Fixation and permeabilization: Fix cells with methanol or paraformaldehyde followed by permeabilization with a detergent if necessary.

• Blocking: Add 2 mL of blocking solution (PBS containing 10% fetal bovine serum) and incubate for 20 minutes at room temperature to reduce non-specific binding .

• Antibody incubation: Remove blocking solution and add 1 mL of PBS/10% FBS containing the FITC-conjugated OBSCN antibody (typically at 1:20-1:200 dilution). Incubate for 1 hour at room temperature in the dark .

• Washing: Wash cells 2 × 5 minutes with PBS to remove unbound antibody .

• Visualization: Observe cells with a fluorescence microscope equipped with a FITC filter (or appropriate filter for excitation at ~495nm and emission at ~525nm) .

How can OBSCN antibody, FITC conjugated be used to study OBSCN's role in cardiac pathologies?

OBSCN has been implicated in cardiac pathologies such as Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). Research using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from ARVC patients with OBSCN mutations has revealed:

• Structural abnormalities: ARVC-iPSC-CMs with OBSCN mutations showed significant lipid accumulation, increased pleomorphism, and irregular Z-bands .

• Calcium current alterations: These cells exhibited increased L-type calcium currents, suggesting a role for OBSCN in calcium handling .

• Protein expression changes: Mutant OBSCN protein and its anchor protein Ank1.5 showed structural disorder and decreased expression, while there was increased expression of junctional protein N-Cadherin .

• Pathway disruptions: Functional enrichment analysis identified pathways involved in focal adhesion, adipocytokines, and PPAR signaling as being altered in the ARVC group .

To study these phenomena, FITC-conjugated OBSCN antibodies can be used in immunofluorescence studies to:

• Visualize OBSCN localization in healthy versus diseased cardiomyocytes

• Examine co-localization with other proteins (using multi-color immunofluorescence)

• Quantify expression levels through fluorescence intensity measurements

What approaches can be used to troubleshoot weak or non-specific signals when using OBSCN antibody, FITC conjugated?

When encountering weak or non-specific signals with OBSCN antibody, FITC conjugated:

• Signal intensity issues:

  • Ensure antibody is stored properly (protected from light)

  • Increase antibody concentration (determine empirically the appropriate dilution)

  • Extend incubation time

  • Optimize fixation protocol (over-fixation can mask epitopes)

  • If necessary, employ signal amplification techniques

• Non-specific binding:

  • Increase blocking time or concentration of blocking agent

  • Add additional washing steps

  • Optimize antibody dilution

  • Use tissues or cells known to express or lack OBSCN as positive and negative controls

  • Consider epitope retrieval techniques for fixed tissues

• Autofluorescence reduction:

  • Include quenching steps to reduce tissue/cell autofluorescence

  • Use appropriate filters to distinguish between FITC signal (emission ~525nm) and autofluorescence

  • Consider longer wavelength fluorophores if autofluorescence is problematic

How can OBSCN antibody, FITC conjugated be used in multi-parameter assays?

For multi-parameter analyses:

• Multi-color immunofluorescence: Combine FITC-conjugated OBSCN antibody with antibodies conjugated to spectrally distinct fluorophores (e.g., TRITC, Cy5) to examine co-localization with other proteins. When designing such experiments, consider:

  • Spectral overlap between fluorophores

  • The order of antibody application (especially important for multi-species primary antibodies)

  • Cross-reactivity between secondary detection systems

• Flow cytometry multi-parameter analysis: FITC-conjugated OBSCN antibody can be combined with other markers to analyze OBSCN expression across different cell populations or under various conditions, as demonstrated in flow cytometry experiments where FITC signal can be specifically quenched to confirm specificity .

• Correlative microscopy: Following immunofluorescence with FITC-conjugated OBSCN antibody, samples can be processed for other imaging modalities (e.g., electron microscopy) to correlate protein localization with ultrastructural features.

How do mutations in the OBSCN gene affect antibody recognition and experimental design?

When studying samples with OBSCN mutations:

• Epitope consideration: Verify whether the mutation affects the epitope recognized by the antibody. OBSCN antibodies may target different regions of this large protein, including C-terminal regions, Rho-GEF domains, or kinase domains .

• Expression level variability: ARVC-iPSC-CMs with OBSCN mutations showed reduced expression of both OBSCN and ANK1.5 genes , which may necessitate optimization of antibody concentration or detection methods.

• Structural alterations: Mutations may cause protein misfolding or altered interaction with binding partners, potentially masking epitopes or changing subcellular localization. This requires careful interpretation of immunofluorescence patterns.

• Control selection: When comparing wild-type and mutant OBSCN, ensure proper controls from the same genetic background where possible to accurately attribute differences to the mutation rather than other variables.

What are the considerations for analyzing OBSCN in different tissue and cell types?

OBSCN expression and function varies across tissues and cell types:

• Tissue-specific expression patterns: While originally characterized in striated muscles, OBSCN expression has been detected in various tissues. Researchers should validate antibody performance in their specific tissue of interest.

• Fixation optimization: Different tissues may require specific fixation protocols to preserve OBSCN epitopes while maintaining tissue morphology.

• Autofluorescence management: Tissues with high autofluorescence (e.g., cardiac tissue) may require special quenching steps or alternative detection strategies.

• Background reduction: Non-specific binding profiles differ between tissues; optimization of blocking and washing steps should be tissue-specific.

• Epitope accessibility: In dense tissues, permeabilization and antigen retrieval methods may need optimization to ensure antibody access to OBSCN epitopes.

How can FITC-conjugated OBSCN antibody results be quantified and validated?

For quantitative analysis and validation:

• Fluorescence intensity quantification:

  • Use appropriate imaging software to measure mean fluorescence intensity

  • Include reference standards for normalization between experiments

  • Apply consistent exposure settings across all samples

• Validation approaches:

  • Confirm findings using alternative detection methods (e.g., Western blotting with non-FITC OBSCN antibodies)

  • Perform gene knockdown or knockout experiments to verify specificity

  • Use multiple antibodies targeting different OBSCN epitopes to confirm results

• F/P molar ratio determination:

  • The Fluorescein/Protein (F/P) molar ratio of the conjugate will vary between different preparations and must be determined empirically

  • This ratio affects signal intensity and potentially background levels

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design

  • Include sufficient biological and technical replicates

  • Consider using automated, unbiased image analysis algorithms to reduce experimenter bias

How can OBSCN antibody, FITC conjugated contribute to iPSC-based disease modeling?

OBSCN antibody, FITC conjugated can significantly enhance iPSC-based disease modeling:

• Cardiomyopathy models: As demonstrated in studies of ARVC-iPSC-CMs with OBSCN mutations, immunofluorescence using OBSCN antibodies can reveal structural abnormalities and altered protein localization patterns .

• Developmental studies: Track OBSCN expression and localization during differentiation of iPSCs into specialized cell types, particularly cardiomyocytes.

• Drug screening applications: Monitor changes in OBSCN expression, localization, or interaction networks in response to therapeutic candidates.

• Personalized medicine approaches: Compare OBSCN characteristics in iPSC-derived cells from patients with different mutations or clinical presentations to understand genotype-phenotype correlations.

What are the considerations for using OBSCN antibody, FITC conjugated in super-resolution microscopy?

When applying super-resolution techniques:

• Photostability optimization: FITC has moderate photostability; consider using oxygen scavenger systems to reduce photobleaching during extended imaging sessions.

• Labeling density: For techniques like STORM or PALM, ensure appropriate labeling density of FITC-conjugated antibodies.

• Secondary detection options: For some super-resolution methods, using primary OBSCN antibody followed by secondary anti-FITC antibodies may provide signal amplification .

• Multi-color super-resolution: When combining with other fluorophores, consider chromatic aberration correction and registration between channels.

• Sample preparation refinement: Super-resolution techniques often require specialized sample preparation to minimize background and maximize signal precision.