CSRP3 Antibody

What is CSRP3 and why is it significant in cardiovascular research?

CSRP3 (Cysteine and glycine-rich protein 3), also known as Muscle LIM Protein (MLP) or Cardiac LIM Protein (CLP), functions as a positive regulator of myogenesis with critical roles in cardiac development and pathology. This 21 kDa protein acts as a cofactor for myogenic bHLH transcription factors including MYOD1, MYOG, and MYF6, enhancing their DNA-binding activity . CSRP3 plays a crucial and specific role in cardiomyocyte cytosolic structure organization, particularly at Z-line structures where it serves as a scaffold protein promoting the assembly of interacting proteins . It's essential for calcineurin anchorage to the Z-line and required for stress-induced calcineurin-NFAT activation . The protein's significance extends to mechanical stretch sensing and maintenance of muscle cell integrity through actin-based mechanisms, as it can directly bind to actin filaments and cross-link them into bundles . Notably, mutations in CSRP3 have been implicated in both dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), making it a valuable target for cardiovascular pathology research .

What criteria should researchers consider when selecting a CSRP3 antibody for their experiments?

When selecting a CSRP3 antibody, researchers should consider multiple technical parameters to ensure experimental success:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IP, etc.) with published literature support if possible .

• Species reactivity: Confirm reactivity with your experimental model organism. Current CSRP3 antibodies show validated reactivity with human, mouse, and rat samples, with predicted reactivity in bovine (97% homology) and porcine (98% homology) models .

• Antibody type: Consider whether a monoclonal antibody (higher specificity, like EPR12615(B) ) or polyclonal antibody (potentially higher sensitivity, like 10721-1-AP ) better suits your research question.

• Epitope location: For functional studies, select antibodies targeting domains relevant to your research question (N-terminal, central region, C-terminal) .

• Detection system compatibility: Ensure secondary detection reagents match your primary antibody host species (rabbit IgG for many available CSRP3 antibodies) .

• Validation data relevance: Examine the manufacturer's validation data specifically in cardiac/muscle tissue, where CSRP3 is predominantly expressed .

• Format considerations: Some applications may benefit from conjugated antibodies, while others require unconjugated formats with appropriate buffer conditions .

How are CSRP3 expression patterns distributed across different tissues?

CSRP3 exhibits a highly tissue-specific expression pattern primarily concentrated in striated muscle tissues:

This highly specific distribution pattern makes CSRP3 particularly valuable as a cardiac-specific marker. When performing experiments with CSRP3 antibodies, researchers should include appropriate positive controls (heart/skeletal muscle) and negative controls (lung/tonsil) to validate antibody specificity . The unexpected detection in pancreatic cancer tissue suggests potential ectopic expression in certain pathological conditions, warranting further investigation in cancer research contexts .

What are the optimal protocols for using CSRP3 antibodies in Western blot applications?

For optimal Western blot results with CSRP3 antibodies, researchers should implement this methodological approach:

Sample Preparation:

• Use fresh tissue lysates from heart or skeletal muscle when possible

• Load appropriate amounts (typically 10-20 μg of total protein)

• Include both positive controls (heart tissue) and negative controls

Protocol Optimization:

• Antibody Dilution: For monoclonal antibodies like EPR12615(B), use 1:1000 dilution; for polyclonal antibodies like 10721-1-AP, test a range from 1:500-1:3000

• Protein Detection: CSRP3 has a predicted molecular weight of 21 kDa, which should be confirmed on blots

• Membrane Transfer: Use PVDF membranes for optimal protein retention, with recommended transfer conditions of 100V for 60-90 minutes in cold transfer buffer containing 20% methanol

• Blocking Conditions: 5% non-fat dry milk in TBST for 1 hour at room temperature typically provides optimal blocking

• Secondary Antibody: For rabbit primary antibodies, use HRP-conjugated anti-rabbit IgG at 1:2000-1:5000 dilution

• Signal Development: Both ECL and fluorescent detection systems are compatible, with exposure times requiring optimization based on expression levels

• Troubleshooting: If observing multiple bands, evaluate potential isoforms (CSRP3 has at least two known isoforms) or post-translational modifications

How should researchers optimize immunohistochemistry protocols for CSRP3 detection in cardiac tissues?

Immunohistochemical detection of CSRP3 in cardiac tissues requires specific optimization steps for reliable results:

Tissue Processing:

• Formalin-fixed paraffin-embedded (FFPE) tissues are suitable for CSRP3 detection

• Section thickness of 4-5 μm is recommended for optimal antibody penetration and signal clarity

Antigen Retrieval:

• Heat-mediated antigen retrieval is critical for CSRP3 detection

• Use pressure cooker method with either:

  • TE buffer at pH 9.0 (preferred for polyclonal antibody 10721-1-AP)

  • Citrate buffer at pH 6.0 (alternative option)

• Complete antigen retrieval before continuing with IHC protocol

Antibody Incubation:

• For monoclonal antibodies like EPR12615(B), use 1:1000 dilution

• For polyclonal antibodies like 10721-1-AP, test dilutions between 1:20-1:200

• Incubate overnight at 4°C for optimal sensitivity and specificity

Detection Systems:

• Both ABC (Avidin-Biotin Complex) and polymer-based detection systems are suitable

• DAB (3,3'-diaminobenzidine) chromogen provides good visualization of CSRP3 expression

Controls and Interpretation:

• Include positive control sections (normal heart tissue)

• Include negative controls (lung or tonsil tissue, or primary antibody omission)

• CSRP3 shows both cytoplasmic and nuclear localization, with predominant cytoplasmic signal in normal cardiac myocytes

Technical Considerations:

• Counterstain with hematoxylin after DAB development

• Mount with permanent mounting medium for long-term storage and analysis

What approaches can be used to study CSRP3 protein-protein interactions in cardiovascular research?

Investigating CSRP3 protein-protein interactions requires specialized techniques due to its role as a scaffold protein at Z-line structures. The following methodological approaches are recommended:

Immunoprecipitation (IP):

• Use 0.5-4.0 μg of CSRP3 antibody per 1.0-3.0 mg of total heart tissue lysate

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Confirm successful pull-down by Western blot analysis using CSRP3 antibody at 1:1000 dilution

• This technique has been successfully validated with fetal heart lysates

GST-Pulldown Assays:

• This approach is particularly valuable for studying CSRP3 interactions with α-Actinin and has successfully demonstrated diminished binding due to p.G78V and p.W140C variants

• Express CSRP3 as a GST-fusion protein in bacterial systems

• Purify using glutathione-agarose beads

• Incubate with cardiac cell lysates to capture interacting proteins

• Analyze bound proteins by Western blot or mass spectrometry

Co-localization Studies:

• Perform dual immunofluorescence staining with antibodies against CSRP3 and potential binding partners

• Use confocal microscopy to visualize co-localization patterns

• This technique has revealed both cytoplasmic and nuclear localization of wild-type CSRP3, with altered localization in mutant forms

Proximity Ligation Assay (PLA):

• This advanced technique can visualize protein interactions in situ

• Requires antibodies from different species against CSRP3 and potential interacting partners

• Provides spatial resolution of interactions within cardiac tissues

Yeast Two-Hybrid Screening:

• Useful for identifying novel CSRP3 interacting partners

• Construct bait plasmids containing CSRP3 coding sequences

• Screen against cardiac cDNA libraries

FRET Analysis:

• For live-cell interaction studies of CSRP3 with potential binding partners

• Requires fluorescent protein tagging of both CSRP3 and interacting proteins

• Provides dynamic temporal data on interactions

How can CSRP3 antibodies be utilized to investigate cardiomyopathy mechanisms?

CSRP3 antibodies serve as powerful tools for investigating cardiomyopathy mechanisms through multiple experimental approaches:

Mutant Protein Expression Analysis:

• Western blot analysis using CSRP3 antibodies can quantify protein expression levels in cardiac tissue from patients with DCM or HCM compared to controls

• Reduced CSRP3 protein levels have been observed in human failing hearts with both dilated and ischemic cardiomyopathy

• Comparing expression of wild-type versus mutant CSRP3 in cellular models helps determine if mutations affect protein stability

Localization Studies in Disease Models:

• Immunohistochemistry with CSRP3 antibodies in cardiac biopsies reveals alterations in protein localization associated with specific mutations

• The p.G78V and p.W140C variants cause significant reduction in cytoplasmic expression of CSRP3 protein, more pronounced with p.W140C mutation

• Dual immunofluorescence can demonstrate altered co-localization with Z-disc proteins in disease states

Cytoskeletal Organization Assessment:

• CSRP3 antibodies can reveal disarrayed actin cytoskeleton in cardiomyocytes expressing mutant forms

• This provides mechanistic insights into how CSRP3 mutations may compromise cardiac structural integrity

Downstream Signaling Analysis:

• Immunoprecipitation with CSRP3 antibodies followed by analysis of binding partners can reveal altered interactions in disease states

• CSRP3 mutations affect binding to α-Actinin, as demonstrated by GST-pulldown assays

• Expression analysis of downstream target genes (Ldb3, Myoz2, Tcap, Tnni3, Ttn) reveals regulatory effects of CSRP3 variants

Transgenic/Knockout Model Validation:

• CSRP3 antibodies are essential for validating genetically modified animal models, confirming absence of protein in knockout models or expression of mutant forms in knock-in models

• Such validation is critical when studying phenotypes like the DCM features observed in CSRP3-deficient mice or HCM features in W4R or C58G point mutation models

What are the critical considerations when analyzing CSRP3 protein isoforms?

Analysis of CSRP3 protein isoforms requires careful experimental design and interpretation:

Isoform Identification:

• CSRP3 has at least two documented isoforms with distinct functional properties

• Western blot analysis should be optimized to resolve these isoforms through extended gel run times or gradient gels

• Antibody selection is crucial, as some antibodies may preferentially detect specific isoforms depending on epitope location

Functional Distinctions:

• Isoform 1 (full-length CSRP3) is the predominant form in adult cardiac tissue

• Isoform 2 may play a role in early sarcomere organization but can negatively regulate myotube differentiation when overexpressed

• Interaction studies indicate isoform 2 can associate with isoform 1, potentially altering CSRP3:CFL2 stoichiometry and downregulating CFL2-mediated F-actin depolymerization

Tissue-Specific Expression Patterns:

• Differential expression analysis across tissues and developmental stages requires antibodies capable of distinguishing between isoforms

• Quantitative Western blot analysis should include appropriate controls for each isoform

Disease-Associated Changes:

• Changes in isoform ratios may occur in pathological conditions

• Analysis of cardiac samples from patients with cardiomyopathies should assess potential alterations in isoform expression patterns

Technical Challenges and Solutions:

• Closely migrating isoforms may require high-resolution electrophoresis techniques

• Verification of isoform identity through immunoprecipitation followed by mass spectrometry provides definitive identification

• Use of isoform-specific antibodies, when available, can simplify analysis

How do CSRP3 mutations impact protein detection using antibodies?

CSRP3 mutations can significantly impact protein detection, requiring careful consideration in experimental design:

Epitope Accessibility Changes:

• Mutations may alter protein folding, potentially masking or exposing epitopes

• Different antibodies targeting distinct regions of CSRP3 should be tested when working with known mutant forms

• For the well-studied W4R mutation, N-terminal-targeting antibodies may show reduced binding efficacy

Protein Expression Level Variations:

• Mutations like p.G78V and p.W140C have been shown to reduce CSRP3 protein levels in experimental models

• Quantitative Western blot analysis should be calibrated with appropriate loading controls and reference standards

• Longer exposure times may be necessary to detect reduced protein levels, while avoiding signal saturation for controls

Subcellular Localization Alterations:

• Immunostaining demonstrates that while wild-type CSRP3 shows both cytoplasmic and nuclear localization, variants p.G78V and p.W140C cause obvious reduction in cytoplasmic expression

• Confocal microscopy with Z-stack acquisition may be necessary to fully characterize altered localization patterns

• Co-staining with organelle markers can provide context for altered localization

Protein-Protein Interaction Disruptions:

• GST-pulldown assays demonstrate diminished binding of mutant CSRP3 (p.G78V and p.W140C) with α-Actinin

• Immunoprecipitation efficiency may be reduced when using antibodies with epitopes near mutation sites

• Cross-linking prior to immunoprecipitation may help preserve transient or weakened interactions

Recommendations for Mutation Analysis:

• Use multiple antibodies targeting different epitopes when studying mutant forms

• Include wild-type controls processed in parallel for direct comparison

• Consider complementary approaches such as epitope tagging when antibody detection is compromised by mutations

How can researchers troubleshoot common issues when using CSRP3 antibodies?

When encountering challenges with CSRP3 antibody experiments, researchers should implement systematic troubleshooting strategies:

Western Blot Issues:

• No signal or weak signal:

  • Verify CSRP3 expression in your sample (heart/skeletal muscle positive; lung/tonsil negative)

  • Increase antibody concentration (try 1:500 for polyclonal antibodies)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Enhance detection sensitivity with amplified chemiluminescence substrates

  • Verify transfer efficiency with reversible protein stains

• Multiple bands or unexpected molecular weight:

  • Consider isoforms (CSRP3 has at least two documented isoforms)

  • Evaluate potential post-translational modifications

  • Increase wash stringency to reduce non-specific binding

  • Test alternative antibodies targeting different epitopes

Immunohistochemistry Challenges:

• Poor or absent staining:

  • Optimize antigen retrieval (pressure cooker method is recommended)

  • Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) for retrieval

  • Increase antibody concentration (1:20-1:50 for initial optimization)

  • Extend primary antibody incubation (overnight at 4°C)

  • Verify fixation compatibility (overfixation can mask epitopes)

• High background or non-specific staining:

  • Extend blocking times (2 hours at room temperature)

  • Use protein-free blockers if protein cross-reactivity is suspected

  • Increase wash buffer detergent concentration slightly

  • Titrate primary antibody to optimal concentration

  • Ensure tissue sections are not dried during protocol

Immunoprecipitation Problems:

• Failed IP or low yield:

  • Increase antibody amount (up to 4.0 μg per reaction)

  • Pre-clear lysates more thoroughly

  • Extend antibody-lysate incubation time (overnight at 4°C)

  • Verify lysis buffer compatibility with epitope accessibility

  • Use gentler elution conditions to preserve antibody-antigen interactions

• Non-specific co-immunoprecipitated proteins:

  • Increase wash stringency (higher salt concentration)

  • Add detergents to wash buffers (0.1% NP-40)

  • Use pure recombinant proteins as controls to verify specificity

What quality control measures should be implemented when using CSRP3 antibodies?

Rigorous quality control is essential for generating reliable data with CSRP3 antibodies:

Antibody Validation Controls:

• Positive and negative tissue controls:

  • Positive: Heart tissue, skeletal muscle tissue

  • Negative: Lung tissue, tonsil tissue

  • These tissue controls should be processed alongside experimental samples

• Recombinant protein controls:

  • Use purified CSRP3 protein at known concentrations for Western blot standards

  • Create standard curves for quantitative analyses

• Genetic validation:

  • When possible, include CSRP3 knockout samples as negative controls

  • Alternatively, use siRNA/shRNA knockdown samples with verified reduction in CSRP3 expression

Experimental Design Controls:

• Technical replicates:

  • Minimum of three technical replicates per experiment

  • Consistent loading controls for Western blots (GAPDH, β-actin, total protein staining)

• Antibody specificity controls:

  • Primary antibody omission controls

  • Isotype controls at equivalent concentration to primary antibody

  • Blocking peptide competition assays to confirm specificity

• Cross-validation approaches:

  • Use multiple antibodies targeting different epitopes

  • Validate key findings with orthogonal techniques (e.g., mass spectrometry)

Documentation and Reproducibility:

• Detailed antibody information reporting:

  • Catalog number and lot number

  • Host species and clonality

  • Epitope information when available

  • Dilution and incubation conditions

• Protocol standardization:

  • Maintain detailed protocols with all parameters specified

  • Record any deviations or optimizations

• Image acquisition standardization:

  • Consistent exposure settings for Western blot imaging

  • Standardized microscope settings for immunohistochemistry/immunofluorescence

How should researchers address conflicting data from different CSRP3 antibodies?

When faced with conflicting results from different CSRP3 antibodies, researchers should implement a systematic resolution approach:

Characterize Antibody Differences:

• Epitope mapping:

  • Determine precise epitope locations for each antibody

  • Assess whether epitopes might be differentially affected by protein conformation or interactions

  • Review literature for known post-translational modifications near epitope regions

• Antibody format comparison:

  • Document differences in antibody formats (monoclonal vs. polyclonal)

  • Note species of origin and production methods

  • Consider potential lot-to-lot variations, especially for polyclonal antibodies

Experimental Validation:

• Side-by-side comparison:

  • Test all antibodies simultaneously under identical conditions

  • Include gradient dilution series to rule out concentration effects

  • Process all samples with standardized protocols

• Orthogonal validation:

  • Verify key findings with non-antibody techniques where possible

  • Consider mass spectrometry for protein identification

  • Use genetic approaches (overexpression, knockdown) to validate specificity

• Literature cross-referencing:

  • Thoroughly review published studies using each antibody

  • Identify consensus findings across multiple antibodies

  • Note discrepancies and methodological differences that might explain them

Resolution Strategies:

• For conflicting localization data:

  • Perform co-localization studies with established markers

  • Use subcellular fractionation followed by Western blot to biochemically verify localization

  • Consider live-cell imaging with fluorescently tagged CSRP3 as an antibody-independent approach

• For conflicting protein interaction data:

  • Perform reciprocal immunoprecipitations

  • Use proximity ligation assays for in situ interaction verification

  • Consider yeast two-hybrid or similar systems as antibody-independent validation

• For conflicting expression level data:

  • Correlate protein levels with mRNA expression

  • Use absolute quantification with purified standards

  • Implement targeted mass spectrometry for precise quantification

Reporting Discrepancies:

• Transparent communication:

  • Clearly document all conflicting results in publications

  • Discuss potential reasons for discrepancies

  • Avoid selectively reporting only concordant results

• Methodology emphasis:

  • Provide complete methodological details to allow reproduction

  • Specify exact antibody dilutions, incubation conditions, and detection methods

What emerging techniques might enhance CSRP3 research beyond traditional antibody applications?

Emerging technologies offer promising avenues to advance CSRP3 research beyond conventional antibody-based approaches:

CRISPR-Based Approaches:

• CRISPR/Cas9 genome editing enables precise modification of endogenous CSRP3

• Creation of knock-in cell lines with fluorescent protein tags for live imaging

• Introduction of specific cardiomyopathy-associated mutations in cellular and animal models

• CRISPRi/CRISPRa systems for controlled modulation of CSRP3 expression

Advanced Imaging Technologies:

• Super-resolution microscopy (STORM, PALM, SIM) for detailed visualization of CSRP3 localization at the Z-disc with nanometer precision

• Lattice light-sheet microscopy for dynamic 3D visualization of CSRP3 in living cardiomyocytes

• Expansion microscopy to physically enlarge cellular structures for improved visualization of CSRP3 within the complex sarcomere architecture

Proximity Labeling Approaches:

• BioID or APEX2 fusion proteins to identify proximal proteins in living cells

• This approach can map the comprehensive CSRP3 interactome in different subcellular compartments

• Particularly valuable for identifying transient or weak interactions missed by traditional immunoprecipitation

Mass Spectrometry Innovations:

• Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for precise CSRP3 quantification

• Cross-linking mass spectrometry (XL-MS) to map CSRP3 structural interactions

• Phosphoproteomics and other post-translational modification analyses to characterize CSRP3 regulation

Single-Cell Technologies:

• Single-cell proteomics to analyze CSRP3 expression heterogeneity across cardiomyocyte populations

• Spatial transcriptomics correlated with CSRP3 protein localization

• Multiomics approaches integrating transcriptomic, proteomic, and functional data at single-cell resolution

In Vitro Modeling Systems:

• Human iPSC-derived cardiomyocytes harboring patient-specific CSRP3 mutations

• Engineered heart tissues (EHTs) for functional assessment of CSRP3 variants

• Organ-on-chip technologies incorporating mechanical stress parameters to study CSRP3's mechanosensing functions

How might CSRP3 antibodies contribute to developing therapeutic approaches for cardiomyopathies?

CSRP3 antibodies can facilitate therapeutic development for cardiomyopathies through multiple research pathways:

Diagnostic Applications:

• Development of standardized immunohistochemical protocols for CSRP3 detection in cardiac biopsies

• Creation of sensitive assays to detect circulating CSRP3 as a potential biomarker for cardiac damage

• Multiplexed immunoassays combining CSRP3 with other cardiac markers for improved diagnostic accuracy

Drug Discovery Screening:

• High-content screening platforms using CSRP3 antibodies to identify compounds that normalize aberrant localization of mutant proteins

• Assays to discover molecules that stabilize mutant CSRP3 protein or enhance its interaction with critical binding partners

• Validation of compounds that can upregulate CSRP3 expression in DCM models where protein levels are reduced

Gene Therapy Validation:

• Assessment of CSRP3 expression following adeno-associated virus (AAV)-mediated gene delivery

• Quantification of wild-type CSRP3 replacement in models carrying pathogenic mutations

• Monitoring spatial distribution of virally-expressed CSRP3 in cardiac tissue

RNA Therapeutic Development:

• Evaluation of antisense oligonucleotide effectiveness in modulating CSRP3 splicing

• Assessment of mRNA therapy approaches for delivering wild-type CSRP3 to affected tissues

• Validation of siRNA strategies targeting specific mutant CSRP3 alleles

Protein-Based Therapeutics:

• Development of peptide mimetics based on CSRP3 binding domains

• Validation of peptide delivery using antibody detection of endogenous interactions

• Assessment of protein replacement therapy approaches

Preclinical Model Assessment:

• Standardized protocols for CSRP3 protein evaluation in animal models of cardiomyopathy

• Correlation of CSRP3 expression/localization with functional cardiac parameters

• Longitudinal studies tracking CSRP3 changes during disease progression and therapeutic intervention

What are the research gaps in understanding CSRP3 function that future antibody development might address?

Several critical knowledge gaps in CSRP3 biology could be addressed through advanced antibody development:

Isoform-Specific Functions:

• Development of highly specific antibodies distinguishing between CSRP3 isoforms

• These would enable detailed characterization of isoform-specific expression patterns across development and disease states

• Current understanding of isoform 2's role in early sarcomere organization and negative regulation of myotube differentiation could be expanded

Post-Translational Modifications:

• Generation of modification-specific antibodies (phospho-CSRP3, acetylated CSRP3, etc.)

• These would facilitate mapping of signaling pathways regulating CSRP3 function

• Particular focus on modifications that might mediate CSRP3's role in cardiac stress signaling and PKC/PRKCA regulation

Conformational States:

• Development of conformation-specific antibodies that distinguish between different structural states of CSRP3

• These could help resolve the conflicting reports regarding CSRP3's role in cytoskeleton dynamics and actin depolymerization

• Potentially address the mechanism by which CSRP3 both enhances and reduces CFL2-mediated F-actin depolymerization under different conditions

Temporal Dynamics:

• Antibodies optimized for live-cell imaging applications

• These would advance understanding of CSRP3 dynamics during sarcomere assembly and adaptation to mechanical stress

• Integration with optogenetic approaches for precise spatiotemporal control

Disease Variant-Specific Detection:

• Custom antibodies recognizing specific cardiomyopathy-associated CSRP3 variants

• These would enable direct detection of mutant proteins in heterozygous settings

• Particular value for studying common mutations like W4R that can manifest as either DCM or HCM phenotypes

Tissue-Specific Complexes:

• Development of antibodies recognizing specific CSRP3-containing protein complexes

• These would help clarify how CSRP3 functions in different cellular compartments (Z-disc, nucleus, cytoplasm)

• Advance understanding of CSRP3's dual structural and signaling roles

Non-Muscle Functions:

• Antibodies with enhanced sensitivity for detecting low-level CSRP3 expression

• These could help investigate potential roles in non-muscle tissues

• Particularly relevant given unexpected CSRP3 detection in pancreatic cancer tissue by immunohistochemistry