lmod3 Antibody

What is LMOD3 and what cellular functions does it perform?

LMOD3 (Leiomodin-3) is a member of the leiomodin family of proteins that plays a critical role in sarcomere organization and muscle function. The protein contains several functional domains, including three actin-binding domains, a tropomyosin domain, a leucine-rich repeat (LRR) domain, and a Wiskott-Aldrich syndrome protein homology 2 domain (WH2) . LMOD3 localizes to the pointed ends of thin filaments where it acts as a catalyst for actin nucleation, which is essential for the organization of sarcomeric thin filaments, particularly in skeletal muscles . Research using knockout mouse models has demonstrated that LMOD3 is specifically required for sarcomere integrity in fast muscle fibers, suggesting tissue-specific functional requirements .

What types of LMOD3 antibodies are available for research applications?

Current commercially available LMOD3 antibodies include rabbit polyclonal antibodies that have been validated for multiple research applications. These antibodies are generated by immunizing rabbits with LMOD3 recombinant proteins . For example, some antibodies are produced using the full-length recombinant LMOD3 protein corresponding to Accession Number BC039202 , while others utilize specific fragments such as the E.coli-derived human LMOD3 recombinant protein (Position: M1-R233) . Researchers should select antibodies based on their validated reactivity with human, mouse, or rat LMOD3, depending on their experimental model system.

What is the molecular weight of LMOD3 and how does this inform antibody validation?

The calculated molecular weight of LMOD3 is 64.914 kDa , though Western blot analysis typically detects the protein at 65-70 kDa . This slight discrepancy between the calculated and observed molecular weights may be attributed to post-translational modifications. Research has identified that two isoforms of LMOD3 may exist due to alternative splicing, with predicted molecular weights of 65 kDa and 40 kDa . When validating an LMOD3 antibody, researchers should confirm detection of bands at these expected molecular weights and be aware that tissue-specific expression patterns might affect detection outcomes.

What are the recommended protocols for using LMOD3 antibody in Western blotting?

For optimal results in Western blotting applications with LMOD3 antibody, the following protocol is recommended:

• Sample preparation: Extract proteins from tissue (preferably heart or skeletal muscle) using standard lysis buffers containing protease inhibitors.

• Protein separation: Load 20-50 μg of total protein per lane on SDS-PAGE (10-12% gel).

• Transfer: Transfer proteins to nitrocellulose or PVDF membrane using standard conditions.

• Blocking: Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute LMOD3 antibody at 1:500-1:5000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash membrane 3 times with TBST, 5 minutes each.

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature.

• Detection: Develop using enhanced chemiluminescence (ECL) substrate.

Expected results include detection of bands at 65-70 kDa, corresponding to LMOD3 protein, with strongest signals in heart and skeletal muscle tissues .

How should LMOD3 antibody be used for immunohistochemistry of muscle tissues?

For immunohistochemical detection of LMOD3 in muscle tissues, consider the following methodology:

• Tissue preparation: Fix tissue samples in 10% neutral buffered formalin and embed in paraffin.

• Sectioning: Cut 4-6 μm thick sections and mount on positively charged slides.

• Deparaffinization and rehydration: Process slides through xylene and graded alcohols.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0).

• Blocking: Block endogenous peroxidase with 3% H₂O₂ and non-specific binding with 5% normal serum.

• Primary antibody: Apply LMOD3 antibody at a dilution of 1:20-1:200 and incubate overnight at 4°C .

• Detection: Use an appropriate detection system (e.g., HRP-polymer and DAB substrate).

• Counterstaining: Counterstain with hematoxylin, dehydrate, and mount.

LMOD3 expression is most prominently detected in heart tissue and skeletal muscle, with particularly strong signals observed in human heart tissue sections . When examining results, note the localization pattern within the sarcomere structure, as LMOD3 should be localized to the pointed ends of thin filaments.

What are the recommended storage conditions for maintaining LMOD3 antibody activity?

To maintain optimal activity of LMOD3 antibodies, adhere to the following storage guidelines:

• Temperature: Store antibody at -20°C as recommended by manufacturers .

• Formulation: Most commercial LMOD3 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 , or similar formulations containing PBS, 0.02% NaN3, 1 mg BSA, and 50% glycerol .

• Aliquoting: Do NOT aliquot the antibody to avoid freeze-thaw cycles that can compromise activity .

• Working dilutions: Prepare working dilutions shortly before use and discard any unused diluted antibody.

• Shelf-life: Antibodies are typically stable for 12 months from the date of receipt when stored properly at -20°C .

• Post-reconstitution storage: After reconstitution, antibodies may be stored at 2-8°C for up to 6 months .

Proper storage is critical for maintaining specificity and sensitivity in experimental applications, particularly for polyclonal antibodies.

How can researchers validate the specificity of LMOD3 antibodies in their experimental systems?

Validation of LMOD3 antibody specificity is crucial for generating reliable experimental data. A comprehensive validation approach should include:

• Positive control tissues: Test the antibody on human heart tissue, human skeletal muscle tissue, mouse heart tissue, and rat skeletal muscle tissue, which all express LMOD3 at detectable levels .

• Knockout validation: If available, use LMOD3-knockout models (such as the Lmod3ᴾᴮ/ᴾᴮ mouse model) as negative controls . Western blotting should show absence of the specific band in knockout samples.

• Immunoprecipitation verification: Perform IP experiments with mouse skeletal muscle tissue lysate (approximately 3600μg total protein) using 4μg of anti-LMOD3 antibody, followed by detection with the same antibody at 1:700 dilution .

• Cross-reactivity assessment: Test reactivity in tissues known not to express LMOD3 significantly to ensure absence of non-specific binding.

• Peptide competition: Pre-incubate the antibody with purified LMOD3 recombinant protein to confirm that binding is eliminated in subsequent applications.

This systematic approach will help confirm that observed signals are specific to LMOD3 protein and not the result of cross-reactivity with other cellular components.

What considerations should researchers take when studying LMOD3 in the context of nemaline myopathy models?

When investigating LMOD3 in nemaline myopathy (NM) research models, researchers should consider:

• Model selection: LMOD3-deficient mice display NM with fast fiber-specific atrophy, providing a valuable model for studying the pathogenesis of LMOD3-related NM . These mice show growth retardation, muscle weakness, and sarcomeric disorganization that recapitulates clinical presentations of human patients with LMOD3 mutations .

• Mutation-specific effects: Different mutations in LMOD3 may affect protein function differently. For example, the G326R mutation disrupts the LRR domain structure, altering the binding interface with actin and destroying the ability to regulate thin filament lengths . Researchers should consider designing experiments that can distinguish between complete loss of LMOD3 and mutation-specific functional defects.

• Tissue specificity: LMOD3 deficiency primarily affects fast-twitch muscle fibers, with minimal effects on slow-twitch fibers . Experimental design should include analysis of different muscle types (e.g., EDL vs. soleus muscles).

• Sarcomeric structure analysis: Assessment of Z-line widening (approximately 50% wider in knockout models) and sarcomere disorganization is critical for evaluating NM phenotypes . Immunofluorescence analysis of α-actinin organization can reveal Z-line streaming, a hallmark of NM .

• Functional readouts: Include measurements of grip strength, motor coordination, body weight, and muscle weights as functional parameters affected in NM models .

• Thin filament length measurement: As LMOD3 regulates actin filament lengths, quantitative assessment of thin filament lengths in sarcomeres should be included in experimental protocols .

How can contradictory results with LMOD3 antibodies be reconciled in experimental systems?

When researchers encounter contradictory results using LMOD3 antibodies, several methodological approaches can help identify and resolve these discrepancies:

• Epitope mapping: Different antibodies may recognize distinct epitopes within LMOD3. Commercial LMOD3 antibodies are generated against different regions of the protein (e.g., full-length protein vs. fragments like M1-R233) . If conflicting results occur, consider using antibodies targeting different epitopes to confirm findings.

• Technical variations in detection methods:

  • Adjust antibody dilutions: Test a range of dilutions (e.g., 1:500-1:5000 for Western blots, 1:20-1:200 for IHC)

  • Modify incubation times and temperatures

  • Compare different detection systems

• Sample preparation effects: Differences in protein extraction methods, fixation protocols for IHC, or antigen retrieval techniques can significantly impact antibody binding efficiency.

• Cross-reactivity with LMOD family members: LMOD3 shares homology with other leiomodin family proteins. Verify that the observed signal is specific to LMOD3 rather than LMOD2, which is also expressed in muscle tissue .

• Isoform-specific detection: Consider the potential detection of different LMOD3 isoforms (65 kDa vs. 40 kDa) , which may be differentially expressed across tissues or developmental stages.

• Validation with orthogonal methods: Confirm antibody-based results using complementary techniques such as mRNA quantification, mass spectrometry, or fluorescent protein tagging.

What is the relationship between LMOD3 and the actin cytoskeleton in striated muscle?

LMOD3 plays a critical role in regulating actin filament dynamics in striated muscle:

• Actin nucleation activity: LMOD3 functions as a catalyst for actin nucleation at the pointed ends of thin filaments, contributing to their assembly and organization in sarcomeres .

• Domain-specific interactions: LMOD3 contains three actin-binding domains, a tropomyosin domain, a leucine-rich repeat domain, and a WH2 domain, which collectively mediate its interactions with actin and other thin filament components .

• Opposite function to tropomodulin: While tropomodulins (Tmods) promote actin filament shortening, leiomodins (Lmods) including LMOD3 promote actin filament elongation. This opposing regulatory activity is essential for maintaining proper thin filament lengths in striated muscle .

• LRR domain significance: The leucine-rich repeat domain of LMOD3 houses its second actin-binding site (ABS2) and is critical for the protein's function. The G326 residue within this domain is particularly important, as the G326R mutation disrupts the domain structure and abolishes LMOD3's ability to regulate actin filament lengths .

• Sarcomere maintenance: LMOD3 is required for maintaining sarcomere integrity, particularly in fast-twitch muscle fibers. In LMOD3-deficient mice, sarcomeres show disorganization with irregular F-actin labeling patterns and widened Z-lines .

Understanding this relationship is crucial for interpreting experimental results when using LMOD3 antibodies to study sarcomere organization and muscle function.

What molecular mechanisms underlie LMOD3-related nemaline myopathy?

The molecular pathogenesis of LMOD3-related nemaline myopathy involves several mechanisms:

• Thin filament dysregulation: LMOD3 mutations disrupt the protein's ability to regulate actin filament dynamics, leading to altered thin filament lengths and organization in sarcomeres . This fundamental defect in the basic contractile unit of muscle leads to muscle weakness.

• Mutation-specific effects:

  • Nonsense/frameshift mutations: Most LMOD3 mutations identified in NM patients are nonsense or frameshift mutations leading to protein truncation and loss of function .

  • Missense mutation G326R: This specific mutation, found in compound heterozygous state with truncating mutations in some patients, disrupts the structure of the LRR domain. Molecular dynamics simulations and circular dichroism studies have demonstrated that this single amino acid change perturbs the domain's structure, altering its interaction with actin and abolishing LMOD3's function in regulating thin filament lengths .

• Fiber-type specificity: LMOD3 deficiency primarily affects fast-twitch muscle fibers, with minimal impact on slow-twitch fibers . This explains the pattern of muscle involvement observed in patients.

• Sarcomeric disorganization: In LMOD3-deficient muscle, sarcomeres show significant structural abnormalities, including:

  • Disorganized and narrower F-actin labeling pattern

  • Widened and disorganized Z-lines (approximately 50% wider than normal)

  • Formation of nemaline bodies (protein aggregates) within muscle fibers

• Failed compensation: Despite the presence of other leiomodin family members (e.g., LMOD2), they cannot compensate for LMOD3 deficiency in fast-twitch fibers, highlighting the unique role of LMOD3 in these specific muscle types .

This mechanistic understanding provides a framework for designing targeted therapeutic approaches for LMOD3-related nemaline myopathy.

What are the key differences between studying LMOD3 in various model systems?

Researchers studying LMOD3 should consider important differences between experimental model systems:

When transitioning between model systems, researchers should validate antibody specificity in each system and adjust experimental protocols accordingly. The fiber-type specificity of LMOD3 function is particularly important to consider, as effects may differ dramatically between fast-twitch (e.g., EDL, TA) and slow-twitch (e.g., soleus) muscles .

How can LMOD3 antibodies be utilized in combination with other tools to study sarcomere assembly?

Advanced multimodal approaches combining LMOD3 antibodies with other techniques can provide deeper insights into sarcomere assembly:

• Dual immunofluorescence strategies:

  • Co-staining with LMOD3 antibodies and phalloidin for F-actin visualization allows assessment of thin filament organization

  • Simultaneous labeling with LMOD3 and α-actinin antibodies enables visualization of LMOD3 localization relative to Z-lines

  • Triple labeling with LMOD3, actin, and tropomodulin antibodies can reveal the dynamic relationship between these proteins at thin filament pointed ends

• Super-resolution microscopy approaches:

  • STORM or PALM imaging with LMOD3 antibodies can precisely map its localization within the sarcomere at nanometer resolution

  • Expansion microscopy can provide enhanced visualization of LMOD3 distribution relative to other sarcomeric components

• Live-cell imaging systems:

  • Using LMOD3 antibody fragments conjugated to cell-penetrating peptides in conjunction with fluorescently tagged actin to monitor real-time dynamics

  • Combining with FRAP (Fluorescence Recovery After Photobleaching) to assess LMOD3 turnover at thin filament pointed ends

• Correlative light and electron microscopy (CLEM):

  • Localizing LMOD3 by immunofluorescence, then examining the same structures by electron microscopy to correlate protein localization with ultrastructural features

  • Particularly useful for studying nemaline bodies in disease models

• Proximity labeling approaches:

  • Using APEX2 or BioID fused to LMOD3 to identify proximal interacting partners during sarcomere assembly

  • Validating these interactions using co-immunoprecipitation with LMOD3 antibodies

These integrated approaches provide comprehensive insights into the dynamic role of LMOD3 in sarcomere assembly and maintenance.

What considerations should be taken when designing gene therapy approaches targeting LMOD3?

When developing gene therapy strategies for LMOD3-related disorders, researchers should consider several critical factors:

Researchers can use LMOD3 antibodies to validate the expression and proper localization of delivered gene products in preclinical models.

How can researchers assess the impact of post-translational modifications on LMOD3 function?

Post-translational modifications (PTMs) may significantly influence LMOD3 function, though this area remains relatively unexplored. Researchers can employ the following approaches to investigate PTMs of LMOD3:

• Identification of potential PTMs:

  • Phosphoproteomic analysis of muscle tissue extracts immunoprecipitated with LMOD3 antibodies

  • Mass spectrometry to identify specific modification sites

  • Bioinformatic prediction of potential PTM sites based on sequence analysis

• Site-specific antibodies:

  • Development of antibodies that specifically recognize modified forms of LMOD3 (e.g., phospho-specific antibodies)

  • Validation of these antibodies using phosphatase treatment controls

  • Application in various muscle states (developing, adult, exercised, diseased) to map dynamic changes

• Functional impact assessment:

  • Site-directed mutagenesis to create non-modifiable variants (e.g., S→A for phosphorylation sites)

  • Expression of these variants in LMOD3-deficient backgrounds to assess functional rescue

  • In vitro actin polymerization assays with modified vs. unmodified LMOD3 to assess impact on nucleation activity

• Regulatory enzyme identification:

  • Co-immunoprecipitation studies with LMOD3 antibodies to identify interacting kinases, phosphatases, or other modifying enzymes

  • Inhibitor studies to assess the impact of specific PTM pathways on LMOD3 function

  • siRNA knockdown of candidate enzymes followed by assessment of LMOD3 modification status

• Physiological context:

  • Comparative analysis of LMOD3 PTMs across development, in different muscle types, and in response to exercise or disease

  • Correlation of modification patterns with functional outcomes such as thin filament length and sarcomere organization

Understanding the PTM landscape of LMOD3 may reveal important regulatory mechanisms and potentially identify novel therapeutic targets for nemaline myopathy.