lrrc39 Antibody

What is LRRC39 and why is it significant in cardiac research?

LRRC39 is a component of the sarcomeric M-band that plays a crucial role in myocyte response to biomechanical stress. It may regulate expression of other M-band proteins via an SRF-dependent pathway and is important for normal contractile function in the heart .

The significance of LRRC39 stems from its involvement in the sarcomeric M-band, which, similar to the Z-disc, has been recognized as a hub for signaling pathways mediating diverse cellular processes including cell growth, differentiation, protein turnover, and gene expression . Beyond mechanical function, M-band proteins like LRRC39 are implicated in mechanotransduction pathways that convert structural and mechanical demands to gene transcription.

What types of LRRC39 antibodies are available for research?

Based on the search results, there are several types of LRRC39 antibodies available:

These antibodies have been validated for various applications including Western blotting and immunohistochemistry, making them suitable for different experimental approaches in studying LRRC39 expression and localization .

Which common applications utilize LRRC39 antibodies?

LRRC39 antibodies are commonly used in:

• Western Blot analysis: For detecting LRRC39 protein (~38.6 kDa) in tissue or cell lysates

• Immunohistochemistry (IHC): For visualizing LRRC39 localization in tissue sections, particularly in skeletal muscle and cardiac tissues

• Immunofluorescence (ICC-IF): For subcellular localization studies

• ELISA: For quantitative detection of LRRC39 protein, similar to other protein detection methods described in comparable research

The choice of application depends on the specific research question, with Western blotting being particularly useful for protein expression studies and IHC for localization within tissues.

How should I optimize Western blot conditions for LRRC39 detection?

For optimal Western blot detection of LRRC39:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for tissue lysate preparation, similar to protocols described for other proteins

• Protein loading: Load 25-50 μg of total protein per lane

• Antibody dilution: Use 1:2000 dilution for monoclonal antibodies like OTI2D9 , and 1:500-1:1000 for polyclonal antibodies

• Blocking: Use 2-5% non-fat milk or BSA in TBS-T (Tris-buffered saline with 0.1% Tween-20)

• Incubation: Primary antibody incubation overnight at 4°C; secondary antibody (HRP-conjugated anti-mouse or anti-rabbit IgG) for 1-2 hours at room temperature

• Expected band size: LRRC39 should appear at approximately 38.6 kDa

• Controls: Include positive control (heart or skeletal muscle tissue) and negative control (tissue known not to express LRRC39)

Ensure uniform transfer by confirming with a protein loading control (β-actin, GAPDH) to normalize LRRC39 expression when performing quantitative analysis.

What are the best practices for immunohistochemistry with LRRC39 antibodies?

For effective immunohistochemistry using LRRC39 antibodies:

• Tissue fixation: Use 4% paraformaldehyde for optimal antigen preservation

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended

• Blocking: 5-10% normal serum (match to secondary antibody host) with 1% BSA in PBS

• Primary antibody: Use polyclonal anti-LRRC39 antibody (e.g., HPA077159) at 0.1-0.5 μg/ml concentration

• Incubation time: Overnight at 4°C for primary antibody

• Detection system: Biotin-streptavidin or polymer-based detection systems

• Counterstaining: Hematoxylin for nuclear visualization

• Positive controls: Include heart and skeletal muscle tissues which show high LRRC39 expression

• Negative controls: Include liver tissue and antibody omission controls

For co-localization studies with other sarcomeric proteins, include double immunostaining with M-band markers like myomesin to confirm specificity of LRRC39 localization at the M-band.

How can I validate the specificity of LRRC39 antibodies?

To ensure antibody specificity:

• Multiple antibody validation: Compare staining patterns using at least two different antibodies targeting different epitopes of LRRC39

• Peptide competition assay: Pre-incubate antibody with immunizing peptide (e.g., PEFIGRFQNL IVLDLSRNTI SEIPPGIGLL TRLQELILSY NKIKTVPKEL SNCASLEKLE LAVNRDICDL PQELSNLL for some LRRC39 antibodies)

• Knockout/knockdown controls: Use LRRC39 gene knockdown samples as negative controls

• Recombinant protein: Test antibody against purified recombinant LRRC39 protein

• Cross-reactivity testing: Test in multiple species if claiming cross-reactivity (human, mouse, rat)

• Expression pattern correlation: Compare antibody staining with known tissue expression patterns of LRRC39 (high in heart and skeletal muscle, low in other tissues)

• Correlation with RNA expression: Compare protein detection with LRRC39 RNA-seq data in the same tissues

Proper validation is critical for ensuring reliable experimental results and avoiding false positives or negatives in your research.

How can I design experiments to study LRRC39's role in mechanotransduction?

To investigate LRRC39's role in mechanotransduction:

• Mechanical stress models:

  • Use cyclic stretch apparatus for cultured cardiomyocytes

  • Apply pressure overload via transverse aortic constriction in mouse models

  • Utilize engineered heart tissues with controlled mechanical loading

• LRRC39 manipulation strategies:

  • CRISPR/Cas9-mediated knockout or knockin

  • siRNA knockdown approach

  • Adenoviral overexpression of wild-type or mutant LRRC39

• Downstream signaling analysis:

  • Assess SRF (Serum Response Factor) nuclear translocation and activity using reporter assays

  • Evaluate expression of SRF target genes

  • Analyze phosphorylation status of mechanosensitive kinases

• Protein-protein interaction studies:

  • Co-immunoprecipitation with other M-band proteins

  • Proximity ligation assays to confirm in situ interactions

  • FRET-based approaches for real-time interaction monitoring

• Functional readouts:

  • Sarcomere organization analysis by super-resolution microscopy

  • Contractile performance measurements (force generation)

  • Ca2+ handling assessment

This experimental approach would provide comprehensive insights into LRRC39's mechanotransduction function, similar to studies conducted for other M-band proteins .

What are the considerations for developing antibody-based therapeutic strategies targeting LRRC39?

While primarily a research question, therapeutic antibody development against LRRC39 would require:

• Target accessibility assessment:

  • Determine if LRRC39 has extracellular domains or if intracellular delivery systems are needed

  • Identify accessible epitopes in the protein structure

• Antibody engineering approaches:

  • Consider antibody format (full IgG, Fab, scFv) based on required tissue penetration

  • Engineer antibodies with cardiac-specific targeting capabilities

  • Apply design of experiments (DOE) methodology for optimizing antibody parameters

• Delivery challenges:

  • Address cardiomyocyte-specific delivery mechanisms

  • Consider the blood-cardiac barrier penetration strategies

  • Evaluate stability and half-life in circulation

• Functional modulation:

  • Determine if the antibody should block, enhance, or modify LRRC39 function

  • Design antibodies that specifically modulate SRF-dependent pathways

• Safety considerations:

  • Evaluate off-target effects in other LRRC-containing proteins

  • Assess cardiac functional impact in pre-clinical models

  • Consider compensatory mechanisms that might emerge

Development would need to follow principles similar to those outlined for therapeutic antibody development, applying robust DOE approaches for optimization of conjugation and formulation parameters .

How can I utilize antibody-specific language models to predict potential non-germline mutations in LRRC39 antibodies?

Recent advances in antibody-specific language models (LMs) can be applied to LRRC39 antibody research:

• Understanding germline bias:

  • Most antibody sequences contain predominantly germline residues with only 15-20 non-germline (NGL) residues outside CDR3s

  • Antibody-specific LMs like Sapiens, AntiBERTy, and AbLang-1 predict germline sequences with high frequency (84.9-87.6%)

• Utilizing advanced LMs for antibody design:

  • Apply models like AbLang-2 which perform better at predicting NGL residues

  • Focus on regions with higher probability of beneficial mutations

  • Consider NGL perplexity metrics when evaluating model predictions

• Experimental validation pipeline:

  • Generate a panel of predicted LRRC39 antibody variants

  • Screen for improved binding affinity, specificity, and stability

  • Validate improvements with multiple assay formats

• Data analysis considerations:

  • Account for model biases when interpreting predictions

  • Combine computational predictions with experimental affinity data

  • Use iterative design-build-test cycles for optimization

This approach leverages computational tools like AbLang-2 that have improved capacity to suggest diverse and valid amino acid substitutions for antibody optimization .

What are common pitfalls in LRRC39 antibody experiments and how can they be addressed?

Common pitfalls and solutions include:

When troubleshooting, follow a systematic approach by changing one variable at a time and including appropriate controls with each experiment.

How do I interpret conflicting results between different LRRC39 antibodies?

When faced with conflicting results:

• Epitope analysis:

  • Determine if antibodies recognize different epitopes of LRRC39

  • Consider potential epitope masking due to protein interactions or post-translational modifications

• Validation assessment:

  • Review validation data for each antibody

  • Check specificity using knockout controls or peptide competition assays

  • Evaluate literature reports of antibody performance

• Methodology differences:

  • Consider if differences arise from sample preparation methods

  • Evaluate fixation, antigen retrieval, or blocking protocols

  • Standardize protocols across antibodies when possible

• Biological interpretations:

  • Consider if results reflect different isoforms or post-translational modifications

  • Evaluate if conflicting results occur in specific tissues or conditions

  • Determine if developmental stages affect antibody binding patterns

• Resolution approaches:

  • Use non-antibody based methods (MS/MS, RNA-seq) for validation

  • Employ additional antibodies from different sources

  • Design definitive experiments focusing on the specific conflict

By systematically evaluating these factors, researchers can resolve conflicts and determine which antibody results most accurately reflect biological reality.

How can I quantify LRRC39 expression levels accurately in tissue samples?

For accurate quantification:

• Western blot quantification:

  • Use graduated standard curves with recombinant LRRC39 protein

  • Apply digital image analysis software (ImageJ, Image Lab)

  • Normalize to appropriate loading controls (GAPDH, β-actin)

  • Include linear dynamic range controls

• ELISA-based quantification:

  • Develop sandwich ELISA using capture and detection antibodies against different LRRC39 epitopes

  • Create standard curves using recombinant LRRC39 (R² values ≥0.97)

  • Process samples similar to protocols described for other proteins (e.g., sonication in RIPA buffer with protease inhibitors)

  • Perform at least triplicate measurements

• Immunohistochemistry quantification:

  • Use digital pathology approaches with machine learning algorithms

  • Apply H-score or Allred scoring systems for semi-quantitative analysis

  • Include reference standards on each slide

  • Validate through correlation with other quantification methods

• RT-qPCR correlation:

  • Compare protein quantification with mRNA levels

  • Account for potential post-transcriptional regulation

  • Consider differences between transcriptional and translational regulation

For all methods, ensure biological replicates (n≥3) and technical replicates to ensure statistical robustness of quantification data.

How might LRRC39 antibodies be applied in single-cell analysis of cardiac tissues?

Emerging applications include:

• Single-cell mass cytometry (CyTOF):

  • Conjugate LRRC39 antibodies with rare earth metals

  • Combine with other cardiac markers for comprehensive phenotyping

  • Analyze heterogeneity in LRRC39 expression across cardiomyocyte subpopulations

• Imaging mass cytometry:

  • Apply metal-tagged LRRC39 antibodies to tissue sections

  • Maintain spatial context while achieving single-cell resolution

  • Correlate LRRC39 expression with tissue microenvironment

• Single-cell Western blotting:

  • Adapt LRRC39 antibody protocols to microfluidic single-cell Western platforms

  • Quantify protein expression heterogeneity in isolated cardiomyocytes

  • Correlate with functional parameters at single-cell level

• Spatial transcriptomics integration:

  • Combine LRRC39 immunofluorescence with spatial transcriptomics

  • Correlate protein expression with transcriptional profiles

  • Map LRRC39 expression in relation to pathological tissue regions

These approaches would enable researchers to understand the heterogeneity of LRRC39 expression and function at unprecedented resolution in normal and diseased cardiac tissues.

What are the potential applications of LRRC39 antibodies in studying cardiac disease mechanisms?

LRRC39 antibodies could advance cardiac disease research through:

• Cardiomyopathy biomarker development:

  • Assess LRRC39 expression changes in different cardiomyopathies

  • Evaluate serum/plasma LRRC39 levels in heart failure patients

  • Correlate with disease progression and treatment response

• Sarcomeric remodeling analysis:

  • Track LRRC39 localization changes during pathological remodeling

  • Evaluate co-localization with other M-band proteins in disease states

  • Assess sarcomeric integrity in various cardiac pathologies

• Mechanotransduction pathway investigation:

  • Study SRF-dependent signaling alterations in heart failure

  • Analyze biomechanical stress responses in pressure/volume overload

  • Investigate LRRC39's role in cardiac fibrosis development

• Therapeutic target validation:

  • Use antibodies to identify functional domains for drug targeting

  • Develop blocking antibodies to modulate LRRC39 function

  • Screen for small molecules that mimic or enhance antibody effects

• Genetic cardiomyopathy models:

  • Characterize LRRC39 expression and localization in genetic cardiomyopathy models

  • Correlate with functional parameters and disease progression

  • Identify potential compensatory mechanisms

These applications could reveal LRRC39's role in cardiac pathophysiology and potentially identify new therapeutic strategies for heart disease.

How can advanced imaging techniques enhance LRRC39 antibody-based research?

Advanced imaging with LRRC39 antibodies offers several opportunities:

• Super-resolution microscopy:

  • Apply STORM or PALM techniques with fluorophore-conjugated LRRC39 antibodies

  • Achieve 10-20 nm resolution of sarcomeric structures

  • Precisely map LRRC39 location within the M-band architecture

• Live-cell imaging approaches:

  • Develop cell-permeable LRRC39 antibody fragments

  • Track dynamic changes in LRRC39 localization during contraction cycles

  • Monitor real-time responses to mechanical stress

• Correlative light and electron microscopy (CLEM):

  • Combine immunofluorescence of LRRC39 with electron microscopy

  • Correlate protein localization with ultrastructural features

  • Achieve molecular resolution within structural context

• Expansion microscopy:

  • Apply physical tissue expansion techniques with LRRC39 immunolabeling

  • Enhance visualization of nanoscale protein distribution

  • Resolve closely associated proteins within sarcomeric structures

• Intravital imaging:

  • Develop methods for in vivo tracking of LRRC39 in animal models

  • Monitor dynamic changes during cardiac remodeling

  • Correlate with functional cardiac parameters

These imaging approaches would provide unprecedented insights into LRRC39's structural organization and dynamic behavior in normal and pathological conditions.