MYH11 Antibody, Biotin conjugated

What is MYH11 and why is it an important research target?

MYH11 (Myosin Heavy Chain 11) is a smooth muscle myosin belonging to the myosin heavy chain family. It functions as a major contractile protein and serves as a subunit of a hexameric protein complex consisting of two heavy chain subunits and two pairs of non-identical light chain subunits. This protein plays a crucial role in converting chemical energy into mechanical energy through ATP hydrolysis, making it essential for smooth muscle contraction and cellular motility. MYH11 is particularly significant in research due to its involvement in various pathological conditions, including its association with acute myeloid leukemia of the M4Eo subtype through chromosomal rearrangements, specifically the pericentric inversion of chromosome 16[inv(16)(p13q22)] . The protein has several synonyms including SMMHC (Smooth Muscle Myosin Heavy Chain) and KIAA0866, with a molecular weight of approximately 227 kDa based on calculations, though it may appear at different sizes on Western blots due to post-translational modifications or alternative splicing .

What are the key considerations when selecting a biotin-conjugated MYH11 antibody?

When selecting a biotin-conjugated MYH11 antibody, researchers should consider several critical factors to ensure experimental success. First, verify the antibody's species reactivity matches your experimental model; available MYH11 antibodies demonstrate reactivity with human and rat tissues, and some with mouse tissues as well . Second, confirm the antibody has been validated for your specific application, whether it be Western blotting, immunohistochemistry, immunocytochemistry, or immunofluorescence. Third, consider the clonality of the antibody—both monoclonal (such as clone 1F11) and polyclonal options are available, each with distinct advantages depending on your research question . Fourth, evaluate the site of immunogen recognition, as some antibodies target the C-terminal region of MYH11 . Finally, assess the biotin conjugation method, particularly whether a spacer is used, as this can significantly impact detection sensitivity. Biotin-SP (with a 6-atom spacer) positions the biotin moiety away from the antibody surface, making it more accessible to streptavidin binding sites and potentially increasing assay sensitivity, especially when used with alkaline phosphatase-conjugated streptavidin .

What applications are biotin-conjugated MYH11 antibodies suitable for?

Biotin-conjugated MYH11 antibodies are versatile reagents suitable for multiple experimental applications in smooth muscle research. These antibodies are particularly well-suited for Western blotting (WB), where they can be used at dilutions ranging from 1:500 to 1:1,000 to detect MYH11 protein expression levels in tissue or cell lysates . For immunohistochemistry on paraffin-embedded sections (IHC-P), these antibodies provide excellent visualization of smooth muscle tissue architecture at working dilutions of 1:50 to 1:200 . In immunocytochemistry (ICC), they effectively label MYH11 in cultured cells at dilutions of 1:50 to 1:200, as demonstrated in validation studies using HeLa cells . Additionally, biotin-conjugated antibodies are valuable for immunofluorescence (IF) applications, allowing for signal amplification through the biotin-streptavidin interaction when combined with fluorophore-conjugated streptavidin . The biotin conjugation enables signal enhancement in all these applications, making these antibodies particularly valuable for detecting low-abundance MYH11 protein or for increasing sensitivity in complex tissue samples where background might otherwise obscure specific signals .

What storage and handling protocols maximize the stability of biotin-conjugated MYH11 antibodies?

For optimal stability and performance of biotin-conjugated MYH11 antibodies, proper storage and handling are essential. Long-term storage should be at -20°C, where most antibodies remain stable for approximately 12 months . For frequent use and short-term storage (up to one month), refrigeration at 4°C is acceptable to avoid repeated freeze-thaw cycles that can degrade antibody performance . Most biotin-conjugated MYH11 antibodies are supplied in stabilizing solutions containing glycerol (typically 50%) which prevents freezing at -20°C and maintains protein stability . Additional stabilizing components often include buffer systems like PBS or TBS (pH 7.2-7.4), proteins such as BSA (0.4-1%), and preservatives like sodium azide (0.02%) or Proclin300 to prevent microbial contamination . When handling these antibodies, minimize exposure to room temperature, avoid repeated freeze-thaw cycles (more than 5 cycles can significantly reduce antibody activity), and always centrifuge briefly before opening vials to collect liquid that may have accumulated on the cap or walls during shipping or storage .

What species reactivity is available for biotin-conjugated MYH11 antibodies?

The available biotin-conjugated MYH11 antibodies demonstrate different species reactivity profiles, which researchers must carefully consider when designing experiments. Based on the current product information, most commercially available biotin-conjugated MYH11 antibodies show reactivity to human MYH11 protein, making them suitable for studies using human tissues, cell lines, or clinical samples . Many of these antibodies also cross-react with rat MYH11, providing options for researchers working with rat models . Some antibodies additionally demonstrate reactivity with mouse samples, though this is less common . When selecting an antibody for multi-species studies, it's important to verify that the same antibody clone has been validated across all target species to ensure consistent epitope recognition and binding affinity. The species reactivity derives from the conservation of MYH11 protein structure across mammalian species, but researchers should always validate the antibody in their specific experimental system, particularly when working with less common research models or when using the antibody in novel applications .

How do different detection systems compare when using biotin-conjugated MYH11 antibodies?

When working with biotin-conjugated MYH11 antibodies, researchers have several detection systems available, each with distinct advantages for specific experimental contexts. The streptavidin-based detection systems offer the highest sensitivity due to streptavidin's exceptional affinity for biotin (Kd ≈ 10^-15 M), which is significantly stronger than most antibody-antigen interactions . These systems can be paired with various reporter molecules, including enzymes (HRP, AP) for colorimetric detection or fluorophores for fluorescence microscopy. The choice of detection system significantly impacts experimental outcomes as demonstrated in comparative studies. For chromogenic applications, streptavidin-HRP systems typically provide excellent signal-to-noise ratios in immunohistochemistry of smooth muscle tissues, while alkaline phosphatase systems show superior sensitivity when used with Biotin-SP conjugated antibodies due to the extended accessibility of the biotin moiety to streptavidin binding sites . For fluorescence applications, streptavidin conjugated to bright, photostable fluorophores enables highly sensitive detection of MYH11 in smooth muscle structures. The signal amplification capability of these systems makes them particularly valuable for detecting low-abundance MYH11 isoforms or for visualizing subtle changes in MYH11 expression across different tissue types or pathological states .

What are the optimal protocols for using biotin-conjugated MYH11 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of MYH11 using biotin-conjugated antibodies, a carefully optimized protocol is essential. Begin with proper tissue fixation, preferably using 10% neutral buffered formalin for 24-48 hours, followed by standard paraffin embedding. Cut sections at 4-6 μm thickness and mount on positively charged slides. After deparaffinization and rehydration, heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes at 95-100°C is recommended to unmask antigenic sites potentially altered during fixation. Endogenous peroxidase activity should be blocked using 3% hydrogen peroxide for 10 minutes, followed by protein blocking with 5% normal serum for 30 minutes to minimize non-specific binding. Apply the biotin-conjugated MYH11 antibody at the optimal dilution of 1:50 to 1:200, as determined through titration experiments, and incubate overnight at 4°C in a humidified chamber . For detection, use streptavidin-HRP followed by DAB (3,3'-diaminobenzidine) substrate for visualization. To address potential background from endogenous biotin in tissues, incorporate an avidin-biotin blocking step prior to antibody application. Counterstain with hematoxylin, dehydrate, and mount with permanent mounting medium. This protocol consistently yields specific labeling of smooth muscle cells in vascular walls, gastrointestinal tract, and other smooth muscle-containing tissues, with minimal background and optimal signal-to-noise ratio .

How can signal amplification be optimized with biotin-conjugated MYH11 antibodies?

Optimizing signal amplification with biotin-conjugated MYH11 antibodies requires strategic implementation of amplification techniques while maintaining specificity. The biotin-streptavidin system intrinsically provides amplification due to streptavidin's tetrameric structure that can bind four biotin molecules, but further enhancement can be achieved through several approaches. The most effective primary strategy involves using a biotin-conjugated antibody with a spacer (Biotin-SP), which positions the biotin moiety away from the antibody surface, dramatically improving accessibility to streptavidin binding sites and enhancing detection sensitivity, particularly with alkaline phosphatase conjugates . For multi-layer amplification, employ a sequential application technique: first apply the primary MYH11 antibody, followed by a biotinylated secondary antibody, then introduce a tertiary layer of labeled streptavidin. This approach can increase signal intensity 4-8 fold compared to direct detection methods . For ultrasensitive detection, implement tyramide signal amplification (TSA), which utilizes the catalytic activity of HRP to generate high-density biotin deposition at the site of the antigen. When optimizing these amplification methods, researchers must carefully titrate reagents and minimize incubation times to prevent non-specific binding and background signal. Additionally, include appropriate positive controls (smooth muscle tissues) and negative controls (skeletal muscle or antibody diluent alone) to accurately assess specific versus non-specific signal amplification .

What are the considerations for multiplexing experiments with biotin-conjugated MYH11 antibodies?

Multiplexing experiments with biotin-conjugated MYH11 antibodies require careful planning to achieve specific co-detection while avoiding technical artifacts. The primary consideration is the inherent limitation of using multiple biotin-conjugated primary antibodies simultaneously, as they would all bind to the same streptavidin detection reagent, making it impossible to distinguish between different target proteins. Instead, researchers should employ one of several strategic approaches. First, use the biotin-conjugated MYH11 antibody in combination with directly labeled antibodies against other targets, ensuring the fluorophores have non-overlapping emission spectra. Second, implement sequential detection, where the biotin-conjugated MYH11 antibody is fully developed and blocked before introducing additional primary antibodies with different detection systems. Third, consider tyramide signal amplification with different fluorophore-conjugated tyramides for each biomarker, allowing multiple biotinylated antibodies to be used sequentially. For chromogenic multiplexing, use biotin-conjugated MYH11 antibody with DAB (brown) followed by alkaline phosphatase-conjugated antibodies with Fast Red or similar substrates (red) for the second target. Additionally, when designing multiplexing experiments, verify that antibodies are raised in different host species to prevent cross-reactivity of secondary detection reagents, and thoroughly validate each antibody individually before combining them to ensure specific labeling patterns are maintained in the multiplex setting .

How can researchers troubleshoot non-specific binding or background issues with biotin-conjugated MYH11 antibodies?

Non-specific binding and background issues with biotin-conjugated MYH11 antibodies can significantly impact experimental interpretation but can be systematically addressed through a structured troubleshooting approach. The most common source of background in biotin-based detection systems is endogenous biotin present in many tissues, particularly in kidney, liver, and adipose tissue. This can be effectively mitigated by implementing an avidin-biotin blocking step prior to primary antibody application . For high background in immunohistochemistry, optimize antigen retrieval conditions, as excessive retrieval can expose non-specific binding sites. Titrate the antibody concentration carefully; working dilutions of 1:50-1:200 are recommended starting points, but optimal concentration must be determined empirically for each application and tissue type . Increase blocking stringency by extending blocking time to 60 minutes and using a combination of normal serum (5-10%) and bovine serum albumin (1-3%). For Western blotting applications with high background, increase washing duration and frequency (5 washes x 5 minutes each) with TBST containing 0.1-0.3% Tween-20 . If membrane binding persists, consider alternative blocking agents such as 5% non-fat dry milk or commercial blocking reagents. For detecting low-abundance MYH11 in tissues with high background, implement a biotin-free detection system using directly labeled streptavidin-polymer detection methods. Always include experimental controls: tissue known to express MYH11 (positive control), tissue not expressing MYH11 (negative control), and antibody diluent alone (reagent control) to differentiate between specific signal and technical artifacts .

What are the clinical and research implications of MYH11 detection in pathological conditions?

MYH11 detection using biotin-conjugated antibodies has significant implications in both clinical diagnostics and basic research of pathological conditions. In clinical contexts, MYH11 antibodies serve as critical tools for identifying smooth muscle differentiation in tumors, particularly leiomyomas, leiomyosarcomas, and myofibroblastic neoplasms, where they can help distinguish between tumors of smooth muscle origin and those derived from other lineages. More significantly, MYH11 has direct pathogenic relevance in acute myeloid leukemia (AML) with the inv(16)(p13q22) chromosomal rearrangement, which produces a chimeric protein consisting of the N-terminal portion of core-binding factor beta fused with the C-terminal region of MYH11 . This CBFB-MYH11 fusion protein acts as an oncogenic driver that disrupts normal hematopoiesis. Biotin-conjugated MYH11 antibodies that specifically recognize the C-terminal portion can be valuable tools for studying this fusion protein in research settings . In cardiovascular research, MYH11 antibodies enable investigation of smooth muscle phenotypic modulation in atherosclerosis, restenosis, and aortic aneurysms. Mutations in the MYH11 gene have been linked to familial thoracic aortic aneurysm and dissection (TAAD), making MYH11 detection relevant for both genetic studies and histopathological evaluation of vascular diseases. For researchers investigating these conditions, biotin-conjugated MYH11 antibodies offer enhanced sensitivity for detecting subtle changes in protein expression or localization that may have pathological significance .

What are the key specifications of available biotin-conjugated MYH11 antibodies?

The following table summarizes the key specifications of currently available biotin-conjugated MYH11 antibodies based on manufacturer data:

This comprehensive comparison highlights the similarities and differences between available antibody formats, enabling researchers to select the most appropriate reagent for their specific experimental requirements .

How do biotin-conjugated MYH11 antibodies compare to other detection formats?

Biotin-conjugated MYH11 antibodies offer distinct advantages and limitations compared to alternative detection formats:

What validation data supports the specificity of biotin-conjugated MYH11 antibodies?

Comprehensive validation data confirms the specificity of biotin-conjugated MYH11 antibodies across multiple applications and experimental systems. Western blot analysis demonstrates that these antibodies recognize MYH11 protein at the expected molecular weight range, with the full-length protein calculated at 227 kDa, though observed bands may vary due to alternative splicing or post-translational modifications . Immunohistochemical validation shows specific labeling of smooth muscle cells in tissues known to express MYH11, including vascular walls, gastrointestinal tract, and uterine myometrium, with absence of staining in skeletal and cardiac muscle that do not express this smooth muscle-specific isoform . Immunocytochemistry studies in cell lines such as HeLa cells demonstrate specific cytoplasmic localization consistent with MYH11's role in the contractile apparatus . Negative controls, including isotype-matched non-specific antibodies and absorption with immunizing peptides, confirm binding specificity. Cross-reactivity testing against other myosin heavy chain family members shows minimal non-specific recognition of related proteins. Manufacturers typically validate these antibodies using both positive control tissues with known MYH11 expression and negative control tissues lacking expression to ensure specificity and high affinity . These validation approaches collectively provide robust evidence for the specificity of biotin-conjugated MYH11 antibodies, making them reliable tools for both research and potential diagnostic applications.

What are the key considerations for successful implementation of biotin-conjugated MYH11 antibodies in research?

Successful implementation of biotin-conjugated MYH11 antibodies in research requires careful attention to several critical factors. First, researchers must select the appropriate antibody format (polyclonal or monoclonal) based on the specific experimental requirements, with monoclonal antibodies offering higher reproducibility and polyclonal antibodies potentially providing better sensitivity for certain applications . Second, proper storage and handling protocols must be followed to maintain antibody integrity, including storage at -20°C for long-term preservation and minimizing freeze-thaw cycles . Third, optimal working dilutions must be empirically determined for each application and experimental system, starting with the manufacturer's recommended ranges (1:50-1:200 for IHC/ICC and 1:500-1:1000 for WB) . Fourth, appropriate controls must be incorporated, including positive and negative tissue controls, as well as technical controls to assess background and non-specific binding . Fifth, researchers should implement proper blocking procedures to minimize background, particularly addressing endogenous biotin in tissue samples . Finally, the detection system should be carefully selected based on the required sensitivity and compatibility with other experimental components . By systematically addressing these considerations, researchers can maximize the specificity, sensitivity, and reproducibility of experiments utilizing biotin-conjugated MYH11 antibodies, leading to more reliable and interpretable results in both basic research and translational studies involving smooth muscle biology and related pathologies .