MYH6 Antibody, FITC conjugated

What is MYH6 and why is it an important target for cardiac research?

MYH6 encodes the alpha-cardiac myosin heavy chain (aMHC), which is a major contractile protein exclusively expressed in the myocardium of the heart. This protein plays a crucial role in muscle fiber contraction and is directly related to the heart's blood pumping ability. MYH6 expression is down-regulated in cardiac myopathy and failing hearts, making it an important biomarker in cardiovascular research. Mutations in the MYH6 gene are linked to several heart diseases including dilated and hypertrophic cardiomyopathy, positioning it as a significant molecule in cardiac disease research with potential clinical value for diagnostics and treatment development .

What species reactivity can be expected with commercially available MYH6 antibodies?

Commercial MYH6 antibodies demonstrate varying species reactivity profiles depending on the manufacturer and specific clone. Based on validated data, many MYH6 antibodies show reactivity with human, mouse, and rat samples. Some specific antibodies have also been cited to work with pig, chicken, and goat tissues. When selecting an antibody for your research, it's important to verify the reactivity with your species of interest. The FITC-conjugated MYH6 antibody (ABIN7160586, amino acids 894-1132) specifically shows reactivity with human samples .

What antigen retrieval methods are recommended for MYH6 antibody in fixed tissue sections?

For optimal results when using MYH6 antibodies on fixed tissue sections, two primary antigen retrieval methods are recommended:

• High-pressure antigen retrieval with citrate buffer (pH 6.0) - This method has been validated for paraffin-embedded human heart and skeletal muscle tissues. After dewaxing and hydration, sections should undergo high-pressure antigen retrieval, followed by blocking with 10% normal goat serum for 30 minutes at room temperature .

• Antigen retrieval with TE buffer (pH 9.0) - This alternative method has shown good results with human and mouse heart tissues. If results are suboptimal, citrate buffer (pH 6.0) can be used as an alternative .

After antigen retrieval, sections should be blocked appropriately before overnight incubation with the primary antibody at 4°C.

How can I minimize background fluorescence when using FITC-conjugated MYH6 antibodies?

To minimize background fluorescence when working with FITC-conjugated MYH6 antibodies, consider implementing the following protocol modifications:

• Optimize blocking conditions: Use 10% normal goat serum for 30 minutes at room temperature before primary antibody incubation. Adding 1% BSA to the antibody diluent can further reduce non-specific binding .

• Adjust antibody dilution: Start with the recommended range (1:50-1:500) but perform a titration to determine the optimal concentration that provides specific signal with minimal background .

• Extend washing steps: Implement additional and longer washing steps with PBS containing 0.05-0.1% Tween-20 after primary and secondary antibody incubations.

• Use appropriate negative controls: Include sections with no primary antibody and ideally a tissue known to be negative for MYH6 expression to assess background levels.

• Consider photobleaching protection: FITC is susceptible to photobleaching, so minimize light exposure during the protocol and use anti-fade mounting media.

• Perform autofluorescence quenching: Treat sections with 0.1% Sudan Black in 70% ethanol for 20 minutes before antibody incubation to reduce tissue autofluorescence, particularly important in heart tissue which contains lipofuscin.

What are the storage conditions for maintaining FITC-conjugated MYH6 antibody activity?

To maintain optimal activity of FITC-conjugated MYH6 antibodies, the following storage conditions should be observed:

• Store at -20°C in the dark to prevent photobleaching of the FITC fluorophore.

• For long-term storage, aliquot the antibody to avoid repeated freeze-thaw cycles, which can damage both the antibody and fluorophore.

• The antibody is stable for at least one year after shipment when stored properly at -20°C.

• Some formulations contain storage buffers such as PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which help maintain stability.

• Smaller volume preparations (20μL) may contain 0.1% BSA as a stabilizer.

• When handling the antibody, minimize exposure to light to prevent photobleaching of the FITC conjugate .

How can I validate the specificity of MYH6 antibody labeling in cardiac tissue?

Validating the specificity of MYH6 antibody labeling requires a multi-faceted approach:

• Positive control tissues: Use mouse or human heart tissue known to express high levels of MYH6. Cardiac muscle tissue should show strong staining, while skeletal muscle can serve as a comparative control .

• Knockout/knockdown validation: Reference published studies using MYH6 knockout or knockdown models. Several publications have validated specific MYH6 antibodies using these approaches .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (in this case, recombinant human MYH6 protein fragments such as amino acids 894-1132) before staining to confirm binding specificity .

• Multiple antibody comparison: Use different MYH6 antibodies targeting distinct epitopes to confirm consistent staining patterns.

• Western blot correlation: Confirm that the antibody detects a band at the expected molecular weight (200-220 kDa observed; 224 kDa calculated) in heart tissue lysates .

• Co-localization studies: Perform dual staining with cardiac-specific markers to confirm appropriate cellular and subcellular localization.

How can MYH6-FITC antibodies be utilized in tracking cardiomyocyte differentiation from stem cells?

MYH6-FITC antibodies can serve as valuable tools for monitoring cardiomyocyte differentiation from stem cells through several approaches:

• Temporal expression analysis: MYH6 is a late-stage cardiac differentiation marker. Using FITC-conjugated MYH6 antibodies allows for real-time monitoring of cardiomyocyte maturation during differentiation protocols through flow cytometry or live-cell imaging.

• Co-staining differentiation stages: Combined with antibodies against early cardiac progenitor markers (e.g., Nkx2.5, GATA4) using different fluorophores, researchers can create a timeline of cardiac lineage specification and maturation.

• FACS-based purification: The FITC conjugate enables fluorescence-activated cell sorting to isolate pure populations of MYH6-expressing cardiomyocytes from heterogeneous differentiated cultures for downstream applications.

• High-content screening: In 96-well format differentiation, MYH6-FITC can be used to quantitatively assess the efficiency of different differentiation protocols or small molecule enhancers.

• Functional correlation studies: Combining MYH6-FITC immunostaining with calcium imaging or electrophysiological measurements can correlate expression levels with functional cardiac properties in developing cardiomyocytes .

What are the critical considerations when using MYH6 antibodies to distinguish between alpha and beta myosin heavy chain isoforms?

Distinguishing between alpha (MYH6) and beta (MYH7) myosin heavy chain isoforms requires careful antibody selection and experimental design:

• Epitope specificity: Select antibodies targeting regions with minimal sequence homology between MYH6 and MYH7. The amino acid regions 894-1132 or 833-867 of MYH6 have been utilized for generating specific antibodies .

• Validation in transition models: Test the antibody in models where MYH6/MYH7 isoform switching is known to occur, such as developmental stages or heart failure models, to confirm selective detection.

• Western blot resolution: Although both isoforms have similar molecular weights (approximately 220 kDa), high-resolution gel systems with extended run times may separate them. Confirm specificity by comparing heart tissues known to predominantly express either isoform.

• Species considerations: The ratio of MYH6/MYH7 varies significantly between species. Adult mouse ventricles predominantly express MYH6, while human ventricles predominantly express MYH7, making species selection critical for proper controls.

• Sample preparation temperature: Maintain samples at low temperatures during preparation as proteolytic degradation can affect epitope recognition differently between isoforms.

• Cross-validation: Use RT-qPCR or mass spectrometry to validate protein expression results, as these methods can more definitively distinguish between the highly homologous isoforms .

How can MYH6-FITC antibodies be optimized for super-resolution microscopy techniques?

Optimizing MYH6-FITC antibodies for super-resolution microscopy requires several technical adjustments:

• Fixation protocol optimization: For techniques like STORM or STED, use paraformaldehyde fixation (4%) followed by permeabilization with Triton X-100 (0.1%) to maintain epitope accessibility while preserving structural integrity.

• Dilution re-calibration: Super-resolution microscopy typically requires higher signal-to-noise ratios than conventional microscopy. Test a narrower dilution range starting at 1:50 and gradually increasing to 1:200 to find the optimal concentration .

• Secondary amplification systems: For improved signal in techniques with lower sensitivity to FITC, consider using biotinylated secondary antibodies followed by streptavidin-FITC for signal amplification.

• Mounting media selection: Use specialized mounting media designed for super-resolution microscopy that contains oxygen scavengers to reduce photobleaching of FITC during extended acquisition times.

• Sample thickness adjustment: For techniques like STED or SIM, prepare thinner tissue sections (5-10 μm) than typically used for conventional immunofluorescence to reduce out-of-focus fluorescence.

• Reference structure co-staining: Include co-staining for Z-disc proteins (such as α-actinin) as reference structures to facilitate interpretation of the sarcomeric organization revealed by super-resolution imaging of MYH6 .

How do MYH6 expression patterns differ between normal hearts and models of cardiac hypertrophy?

MYH6 expression undergoes significant changes in cardiac hypertrophy compared to normal hearts:

• Isoform switching: In normal adult human hearts, MYH6 (α-cardiac myosin heavy chain) represents approximately 10% of the total cardiac myosin, with MYH7 (β-cardiac myosin heavy chain) predominating. During pathological hypertrophy, MYH6 expression is further down-regulated while MYH7 is upregulated, representing a return to the fetal gene program.

• Regional differences: In hypertrophic hearts, MYH6 reduction is often more pronounced in the left ventricle compared to the atria, which maintain higher MYH6 expression levels even during disease progression.

• Temporal dynamics: The decrease in MYH6 expression occurs early in the hypertrophic response and can precede clinical manifestations, making it a potential early biomarker of cardiac stress.

• Correlation with function: The ratio of MYH6/MYH7 expression correlates with contractile performance, as MYH6 has higher ATPase activity and faster contractile properties compared to MYH7. The reduction in MYH6 contributes to the decreased contractility observed in hypertrophic hearts.

• Therapeutic responsiveness: Some therapeutic interventions that improve cardiac function in hypertrophy models partially restore MYH6 expression levels, suggesting it as a potential biomarker for treatment efficacy .

What protocol modifications are necessary when using MYH6-FITC antibodies in analyzing cardiac tissue from dilated cardiomyopathy patients?

When analyzing cardiac tissue from dilated cardiomyopathy patients with MYH6-FITC antibodies, several protocol modifications are necessary:

• Antigen retrieval adjustment: Use high-pressure antigen retrieval with citrate buffer (pH 6.0) with extended retrieval times (15-20 minutes) to overcome potential epitope masking due to tissue fibrosis common in cardiomyopathy samples .

• Enhanced blocking: Implement a dual blocking approach using 10% normal goat serum followed by a 30-minute incubation with 5% BSA to reduce background staining, which can be elevated in diseased tissue due to increased extracellular matrix proteins .

• Antibody concentration: Use the lower end of the dilution range (approximately 1:50) initially, as MYH6 expression is typically reduced in dilated cardiomyopathy, requiring more sensitive detection .

• Counterstaining strategy: Include co-staining for fibrotic markers (such as collagen I or fibronectin) and MYH7 to contextualize the reduced MYH6 expression within the disease pathology.

• Section selection and orientation: Carefully select regions that represent both severely affected and relatively preserved myocardium to assess the heterogeneity of MYH6 expression changes.

• Signal amplification: Consider implementing tyramide signal amplification if standard protocols yield weak signals due to the reduced MYH6 expression in diseased tissue.

• Computer-assisted quantification: Use digital image analysis to quantify the MYH6 signal intensity relative to total tissue area, as visual assessment may not capture subtle changes in expression levels .

How can simultaneous detection of MYH6 mutations and protein expression be performed using antibody-based approaches?

Combining mutation detection with protein expression analysis for MYH6 requires sophisticated methodological approaches:

• Epitope-specific antibodies: Utilize antibodies targeting specific MYH6 mutation sites. For example, if researching a mutation in the 894-1132 amino acid region, use the FITC-conjugated antibody specific to this region to potentially differentiate wild-type from mutant protein expression patterns .

• Proximity ligation assay (PLA): This technique can detect protein-protein interactions or specific protein conformations. By using one antibody against a mutation-specific epitope and another against a common MYH6 domain, researchers can visualize where mutated protein exists within the tissue.

• Combined immunofluorescence and FISH: Perform immunofluorescence with MYH6-FITC antibodies followed by fluorescence in situ hybridization using probes specific to known MYH6 mutations to correlate genotype with protein expression at the single-cell level.

• Flow cytometry with conformation-sensitive antibodies: Some antibodies may recognize conformational changes induced by mutations. Using such antibodies in flow cytometry can allow quantitative assessment of mutant protein levels in isolated cardiomyocytes.

• Mass cytometry (CyTOF) with mutation-specific antibodies: This technique allows simultaneous detection of multiple epitopes, enabling researchers to correlate MYH6 mutations with changes in other cardiac proteins within the same cells.

• Immunoprecipitation followed by sequencing: Use MYH6 antibodies to immunoprecipitate the protein, followed by genetic analysis of the precipitated material to correlate specific mutations with protein expression levels .

What are the relative advantages of FITC-conjugated MYH6 antibodies compared to other conjugates for cardiovascular research?

FITC-conjugated MYH6 antibodies offer several distinct advantages for cardiovascular research compared to other conjugates:

• Spectral compatibility: FITC's emission spectrum (peak ~520 nm) allows for effective multiplexing with red-spectrum fluorophores such as Texas Red or Cy5 for co-staining of other cardiac markers, enabling comprehensive analysis of sarcomeric structure and cardiac pathologies.

• Microscopy equipment compatibility: Most fluorescence microscopes are equipped with FITC filter sets as standard, making this conjugate widely accessible without specialized equipment modifications.

• Signal stability in fixed tissue: Although FITC is more prone to photobleaching than some newer fluorophores, it performs reliably in fixed cardiac tissue sections, particularly when used with anti-fade mounting media.

• Cost-effectiveness: FITC conjugation typically results in more affordable antibodies compared to newer fluorophores like Alexa Fluor dyes, allowing for more extensive experimental designs within budget constraints.

• Well-established protocols: The extensive history of FITC use has resulted in highly optimized protocols specifically for cardiac tissue, which may not be as thoroughly developed for newer conjugates.

• Direct visualization: Unlike enzymatic conjugates (such as HRP) that require substrate development, FITC-conjugated antibodies enable direct visualization, simplifying workflow and reducing potential artifacts from development reactions .

How does the sensitivity of FITC-conjugated MYH6 antibodies compare with unconjugated antibodies in multi-step detection systems?

The sensitivity comparison between direct FITC-conjugated MYH6 antibodies and unconjugated antibodies with multi-step detection reveals important performance differences:

• Signal intensity: Multi-step detection systems using unconjugated primary antibodies followed by labeled secondary antibodies typically offer 2-4 fold higher sensitivity than direct FITC conjugates due to signal amplification from multiple secondary antibodies binding each primary antibody.

• Signal-to-noise ratio: While multi-step detection provides stronger signals, direct FITC conjugates often produce cleaner backgrounds with less non-specific binding, particularly valuable in tissues with high autofluorescence like cardiac muscle.

• Detection threshold: For low-abundance MYH6 expression (as in early differentiation models or diseased tissue), unconjugated antibodies with amplification steps can detect protein levels below the threshold of direct FITC conjugates.

• Protocol complexity and time: FITC-conjugated antibodies enable simpler, faster protocols (often single-day) compared to multi-step approaches that require additional incubation and washing steps (typically 1.5-2 days).

• Epitope accessibility: Direct conjugation may impact antibody binding to certain epitopes, particularly in the 894-1132 amino acid region of MYH6. In such cases, unconjugated antibodies might maintain better epitope recognition.

• Multiplexing capability: While FITC-conjugated antibodies simplify double and triple labeling protocols by eliminating concerns about secondary antibody cross-reactivity, they offer less flexibility for signal balancing compared to multi-step systems .

What are the optimal complementary markers for co-immunostaining with MYH6-FITC antibodies in cardiac development studies?

For comprehensive analysis of cardiac development using MYH6-FITC antibodies, the following complementary markers provide optimal insights when used in co-immunostaining protocols:

• Developmental stage markers:

  • NKX2.5 (nuclear marker) - Early cardiac progenitor marker that precedes MYH6 expression

  • GATA4 (nuclear marker) - Transcription factor essential for cardiomyocyte differentiation

  • Cardiac Troponin T (cTnT) - Sarcomeric protein expressed slightly earlier than MYH6 during cardiomyocyte maturation

• Structural context markers:

  • α-Actinin (Z-disc marker) - Provides structural context to MYH6 localization within the sarcomere

  • Connexin 43 (Cx43) - Gap junction protein marking intercellular connections in maturing cardiomyocytes

  • N-cadherin - Adherens junction protein indicating mechanical coupling between cardiomyocytes

• Isoform transition markers:

  • MYH7 (β-myosin heavy chain) - For analyzing the MYH6/MYH7 isoform switch during development

  • Atrial/ventricular markers (MLC2a/MLC2v) - To distinguish chamber-specific differentiation alongside MYH6 expression

• Functional maturation markers:

  • SERCA2a - Calcium handling protein indicating maturation of excitation-contraction coupling

  • Ryanodine receptor (RyR2) - Another calcium handling protein marking functional maturation