OBSCN Antibody, Biotin conjugated

What is OBSCN protein and why is it important in muscle research?

Obscurin (OBSCN) is a large sarcomeric protein found in striated muscle that plays crucial roles in muscle biology and pathology. It functions as a structural component of striated muscles with involvement in myofibrillogenesis - specifically in the assembly of myosin into sarcomeric A bands . The protein possesses serine/threonine protein kinase activity and can phosphorylate N-cadherin (CDH2) and sodium/potassium-transporting ATPase subunit ATP1B1 . OBSCN is particularly important in research because abnormalities in this protein have been linked to both cardiac and skeletal muscle diseases . The gene encoding obscurin (OBSCN) undergoes extensive alternative splicing during development, with 121 non-overlapping exons making it one of the genes coding for the largest mRNAs in the human genome . Recent studies have also demonstrated connections between truncating variants in the OBSCN gene and hypertrophic cardiomyopathy (HCM), with these variants being significantly more prevalent in HCM patients than controls (odds ratio 3.58, P<0.001) .

What are the key experimental applications for OBSCN Antibody, Biotin conjugated?

The OBSCN Antibody, Biotin conjugated provides researchers with several methodological advantages for studying obscurin in experimental contexts:

• Western Blot Analysis: The biotin conjugation enables sensitive detection of obscurin protein expression levels in muscle tissue samples, with recommended dilutions typically between 1:500 and 1:2000 .

• Immunofluorescence/Immunocytochemistry: For localization studies examining the subcellular distribution of obscurin within muscle cells, including its presence in cytosol, M-band, myofibril, nuclear body, plasma membrane, sarcolemma, and Z disc .

• ELISA Applications: For quantitative assessment of obscurin levels in tissue or cell lysates .

• Sarcomere Organization Studies: Particularly valuable for investigating the role of obscurin in myofibrillogenesis and sarcomere integrity .

• Protein-Protein Interaction Analysis: For examining interactions between obscurin and other muscle proteins during development and in pathological conditions .

The biotin conjugation specifically enhances detection sensitivity and facilitates multiplexing with other antibodies in co-localization studies.

What are the recommended handling and storage conditions for maintaining OBSCN Antibody, Biotin conjugated activity?

For optimal preservation of antibody activity, OBSCN Antibody, Biotin conjugated should be stored at -20°C or -80°C upon receipt to maintain its functional properties . Repeated freeze-thaw cycles should be avoided as they can compromise antibody integrity. The antibody is typically supplied in liquid form, preserved in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 .

When working with the antibody, it should be gently thawed on ice and briefly centrifuged before use. For long-term storage, small working aliquots can be prepared to minimize freeze-thaw cycles. The manufacturer's specifications indicate that the biotin-conjugated format enhances stability while allowing flexible detection options through secondary reagents like streptavidin-HRP or streptavidin-fluorophore conjugates.

How should experimental controls be designed when using OBSCN Antibody, Biotin conjugated?

Proper experimental design with appropriate controls is essential when working with OBSCN Antibody, Biotin conjugated:

Positive Controls:

• Human striated muscle tissue samples (cardiac or skeletal) known to express obscurin

• Cell lines with confirmed obscurin expression

Negative Controls:

• Tissues or cell lines lacking OBSCN expression

• Secondary-only controls (omitting primary antibody) to assess background from streptavidin reagents

• Isotype controls using irrelevant biotin-conjugated rabbit IgG at equivalent concentrations

Additional Control Considerations:

• For experimental validation of antibody specificity, comparative analysis using alternative OBSCN antibodies targeting different epitopes can provide confirmation of true binding

• When studying pathological conditions, parallel analysis of healthy and diseased tissues helps establish baseline expression and localization patterns

• For developmental studies, age-matched controls are crucial given that OBSCN undergoes extensive alternative splicing during human development

What dilution optimization strategies should be employed for different experimental techniques?

When working with biotin-conjugated antibodies, it's crucial to account for endogenous biotin in certain tissues, particularly in liver, kidney, brain, and adipose tissues. Consider biotin blocking steps in your protocol if analyzing these tissues. Additionally, appropriate blocking buffers containing BSA or normal serum compatible with streptavidin detection systems should be employed to minimize non-specific binding.

What considerations should be made when interpreting sarcomeric localization patterns of obscurin?

Interpretation of sarcomeric localization patterns using OBSCN Antibody, Biotin conjugated requires careful consideration of multiple factors:

• Multiple Cellular Locations: Obscurin has been documented in multiple subcellular compartments, including cytosol, M-band, myofibril, nuclear body, plasma membrane, sarcolemma, and Z disc . The specific localization pattern may vary depending on:

  • Muscle type (cardiac vs. skeletal)

  • Developmental stage

  • Physiological or pathological state

• Isoform-Specific Localization: With 121 non-overlapping exons and extensive alternative splicing , different obscurin isoforms may localize to distinct sarcomeric regions. The epitope recognized by the antibody (amino acids 1621-1712 of human OBSCN) should be considered in relation to the isoforms present in the specific tissue being studied.

• Co-localization Analysis: For definitive sarcomeric localization, co-staining with established markers is recommended:

  • M-band: Myomesin

  • Z-disc: α-actinin

  • A-band: Myosin heavy chain

• Contractile State Considerations: The sarcomeric localization of obscurin can be affected by the contractile state of the muscle. In research examining contractile properties, such as diaphragm studies showing impaired contractile performance in Obscn KO models , fixation conditions should be standardized to control for sarcomere length variations.

How can OBSCN Antibody, Biotin conjugated be utilized in investigating muscle pathologies?

The OBSCN Antibody, Biotin conjugated offers significant value in investigating muscle pathologies:

• Hypertrophic Cardiomyopathy (HCM) Research: Given the established association between OBSCN truncating variants and HCM (odds ratio 3.58, P<0.001) , the antibody can be used to examine obscurin expression and localization in cardiac tissue samples from patients with HCM or animal models of the disease. This can help elucidate the molecular mechanisms by which OBSCN mutations contribute to disease pathogenesis.

• Contractile Dysfunction Analysis: Studies have demonstrated that Obscn knockout models exhibit impaired intracellular Ca2+ dynamics and sarcomere function . The antibody can be employed to correlate obscurin expression patterns with functional defects in muscle contractility:

  • In diaphragm models, Obscn KO shows greater force decline during fatigue (0.28 ± 0.03 P40) compared to wild-type (0.50 ± 0.06 P40)

  • Altered force-velocity relationships in Obscn KO muscle with differences in curvature parameters (a/P0 ratio: 0.27 ± 0.03 in KO vs. 0.36 ± 0.03 in WT)

• Developmental Abnormalities: By examining obscurin expression across different developmental stages using the antibody, researchers can identify critical periods where abnormal expression might lead to congenital muscle disorders. The extensive alternative splicing of OBSCN during human development makes developmental studies particularly informative.

• Biomarker Development: The antibody may be utilized in developing diagnostic or prognostic biomarkers for muscle disorders, particularly since OBSCN truncating variants have been associated with increased risk of cardiovascular death (adjusted hazard ratio, 3.1) .

What methodological approaches can address the challenges of detecting large OBSCN isoforms?

Detecting the full range of OBSCN isoforms presents significant technical challenges due to the gene's extensive alternative splicing and large protein products. Researchers can employ the following methodological approaches:

• Isoform-Specific Detection Strategies:

  • Combine OBSCN Antibody, Biotin conjugated with isoform-specific antibodies targeting different domains

  • Design primers for RT-PCR that span unique exon junctions identified through comprehensive splicing analysis

  • Utilize the exon inclusion level data (PSI or Ψ values) measured using tools like the Intron Exon Retention Estimator (IntEREst)

• Specialized Protein Separation Techniques:

  • Employ gradient gels (2-8% or 3-10%) for better resolution of high molecular weight isoforms

  • Use extended electrophoresis times at lower voltages

  • Consider pulse-field gel electrophoresis for very large isoforms

• Advanced Microscopy Methods:

  • Super-resolution microscopy techniques (STED, STORM, SIM) for precise localization of different obscurin domains

  • Live-cell imaging with domain-specific tags to track dynamic localization changes

• Integration with RNA-Seq Data:

  • Correlate protein detection results with RNA-Seq data analyzing OBSCN alternative splicing patterns

  • Consider tissue-specific expression patterns observed in studies examining fetal vs. adult tissues

  • Account for the 126 unique or 121 non-overlapping exons identified in comprehensive studies

How can OBSCN Antibody, Biotin conjugated be integrated into multi-omics approaches for muscle research?

Integration of OBSCN Antibody, Biotin conjugated into multi-omics research approaches can provide comprehensive insights into muscle biology and pathology:

• Proteomics Integration:

  • Immunoprecipitation using biotinylated OBSCN antibody followed by mass spectrometry to identify obscurin-interacting proteins

  • Correlation of obscurin protein levels detected by the antibody with global proteomic profiles

  • Phosphoproteomic analysis to investigate obscurin's role as a kinase and its downstream targets

• Transcriptomics Correlation:

  • Combined analysis with RNA-Seq data to correlate protein expression with transcript levels

  • Integration with alternative splicing analysis, particularly relevant given OBSCN's 121 non-overlapping exons

  • Examination of splicing factor expression in relation to OBSCN exon inclusion patterns

• Genomics Applications:

  • Correlation of OBSCN protein expression and localization with genetic variants, particularly the truncating variants associated with HCM

  • Assessment of genotype-phenotype relationships in patients with OBSCN variants

  • Implementation in functional studies validating the impact of OBSCN variants identified in whole-exome sequencing studies

• Functional Multi-omics:

  • Combining antibody-based protein detection with functional assays measuring muscle contractility

  • Correlating obscurin localization patterns with mathematical models of sarcomere mechanics

  • Integration of calcium handling data with obscurin expression patterns, particularly relevant given the impaired intracellular Ca2+ dynamics observed in Obscn KO models

What are common technical issues when using OBSCN Antibody, Biotin conjugated and their solutions?

For challenging tissues, antigen retrieval optimization is particularly important. This might include exploring different retrieval methods (heat-induced vs. enzymatic) and buffer compositions (citrate vs. EDTA-based) to ensure optimal epitope accessibility while maintaining tissue morphology.

How should researchers address data interpretation challenges when studying OBSCN in disease models?

Interpreting data from OBSCN studies in disease models presents several challenges:

• Distinguishing Causality from Association:

  • While OBSCN truncating variants show association with HCM (odds ratio 3.58) , establishing direct causality requires additional approaches

  • Combination of antibody-based protein studies with functional assays (contractility measurements, calcium handling) helps establish mechanistic links

  • Consider parallel analysis of other sarcomeric proteins to identify primary vs. secondary effects

• Accounting for Isoform Complexity:

  • The extensive alternative splicing of OBSCN means disease-relevant changes might affect specific isoforms

  • Correlate antibody detection results with RNA-level isoform analysis

  • Consider developmental context, as prenatal and postnatal OBSCN splicing patterns differ significantly

• Addressing Sample Heterogeneity:

  • Patient-derived samples show natural variability; increase sample numbers to account for this

  • Standardize tissue collection and preparation protocols

  • Include detailed clinical data correlation for patient samples

• Resolving Contradictory Findings:

  • When antibody-based protein detection results contradict functional data, consider:

    • Technical limitations in detecting specific isoforms

    • Post-translational modifications affecting antibody binding

    • Compensatory mechanisms in model systems

  • For example, in Obscn KO studies showing impaired contractile performance , protein expression data should be interpreted alongside functional measurements

What methodological considerations apply when studying the kinase activity of obscurin?

The OBSCN protein possesses serine/threonine protein kinase activity, which adds complexity to functional studies. When investigating this aspect with OBSCN Antibody, Biotin conjugated, researchers should consider:

• Epitope Location Considerations:

  • The antibody targets amino acids 1621-1712 of human OBSCN , which should be evaluated in relation to the kinase domains

  • Determine whether the antibody binding affects kinase activity in functional assays

• Kinase Activity Assays:

  • In vitro kinase assays using immunoprecipitated obscurin (via the biotinylated antibody)

  • Validation with known substrates (N-cadherin CDH2 and sodium/potassium-transporting ATPase subunit ATP1B1)

  • Phospho-specific antibody detection of substrate phosphorylation

• Physiological Relevance Assessment:

  • Correlation of in vitro kinase activity with functional outcomes in muscle models

  • Investigation of phosphorylation state changes in Obscn KO models showing contractile dysfunction

  • Analysis of potential compensatory kinase activities in OBSCN-deficient models

• Signaling Pathway Integration:

  • Investigation of obscurin's role as both a structural protein and signaling molecule

  • Assessment of how mechanical stress affects kinase activity

  • Exploration of potential mechanotransduction pathways involving obscurin

How might OBSCN Antibody, Biotin conjugated contribute to therapeutic development for muscle disorders?

The OBSCN Antibody, Biotin conjugated could play several roles in therapeutic development:

• Target Validation:

  • Confirmation of obscurin involvement in disease pathogenesis through detection of altered expression or localization

  • Monitoring obscurin levels and distribution in response to experimental therapeutics

  • Validation of gene therapy approaches targeting OBSCN

• Biomarker Development:

  • Potential use in developing diagnostic or prognostic biomarkers, particularly given the association of OBSCN truncating variants with increased cardiovascular death risk (adjusted hazard ratio, 3.1)

  • Monitoring therapy responses through changes in obscurin expression or localization patterns

  • Stratification of patients for clinical trials based on obscurin expression profiles

• Therapeutic Monitoring Applications:

  • Assessment of obscurin restoration in gene therapy approaches

  • Evaluation of compensatory protein expression changes in response to treatments targeting related pathways

  • Monitoring sarcomeric structural integrity during therapeutic interventions

• Personalized Medicine Approaches:

  • Correlation of patient-specific OBSCN variants with protein expression and localization patterns

  • Development of patient-derived models for personalized therapeutic testing

  • Integration with genetic screening data to identify patients most likely to benefit from specific interventions

What emerging technologies might enhance OBSCN research beyond current antibody-based methods?

Several emerging technologies hold promise for advancing OBSCN research beyond traditional antibody-based approaches:

• CRISPR-Based Technologies:

  • CRISPR-mediated tagging of endogenous OBSCN for live-cell imaging

  • Domain-specific editing to investigate function of specific regions

  • Base editing for modeling disease-relevant mutations

• Advanced Imaging Methods:

  • Super-resolution microscopy for precise localization within sarcomeric structures

  • Label-free imaging technologies (SRS, CARS) to avoid potential artifacts from antibody binding

  • Intravital microscopy for studying obscurin dynamics in living systems

• Single-Cell Analysis:

  • Single-cell proteomics to detect cell-to-cell variability in obscurin expression

  • Integration with single-cell transcriptomics to correlate with splicing patterns

  • Spatial transcriptomics to map regional variations in OBSCN expression within tissues

• Biomechanical Approaches:

  • Atomic force microscopy to assess the contribution of obscurin to sarcomere mechanical properties

  • Traction force microscopy to correlate obscurin expression with cellular contractile forces

  • Microfluidic devices for high-throughput mechanical phenotyping of muscle cells with varying obscurin levels

• Computational Modeling:

  • Molecular dynamics simulations to understand obscurin's structural roles

  • Machine learning approaches to predict functional consequences of OBSCN variants

  • Systems biology models integrating obscurin into larger networks of muscle function

How can researchers integrate OBSCN studies with other sarcomeric protein investigations?

Effective integration of OBSCN research with studies of other sarcomeric proteins requires systematic approaches:

• Co-Localization Studies:

  • Multiplex immunofluorescence combining OBSCN Antibody, Biotin conjugated with antibodies against other sarcomeric proteins

  • Super-resolution co-localization analysis to determine precise spatial relationships within sarcomeric structures

  • Live-cell imaging to track dynamic interactions during sarcomere assembly and contraction cycles

• Protein-Protein Interaction Networks:

  • Proximity labeling approaches (BioID, APEX) using obscurin as bait to identify interaction partners

  • Co-immunoprecipitation studies followed by mass spectrometry

  • Yeast two-hybrid or mammalian two-hybrid screening to identify direct interaction partners

• Functional Redundancy Assessment:

  • Comparative analysis of phenotypes in single vs. combined knockdown/knockout models

  • Rescue experiments to determine if other proteins can compensate for obscurin deficiency

  • Expression profiling to identify compensatory upregulation of related proteins

• Integrated Disease Modeling:

  • Correlation of OBSCN variants with other sarcomeric gene variants in patients with cardiomyopathies

  • Development of multi-gene models to better recapitulate complex disease phenotypes

  • Systems-level analysis of sarcomeric protein networks in health and disease

Through these integrated approaches, researchers can place obscurin in its proper context within the complex architecture and function of the sarcomere, potentially revealing new therapeutic targets and diagnostic approaches for muscle disorders.