CASQ1 Antibody

Basic Research Questions

• What is CASQ1 and what roles does it play in muscle physiology?

CASQ1 is a high-capacity, low-affinity calcium-binding protein primarily expressed in skeletal muscle sarcoplasmic reticulum. It functions as an active participant in calcium movement and distribution by interacting with stromal interaction molecule 2 (STIM2) as well as participating in calcium sensing and buffering mechanisms . The protein contains specific high-affinity Ca²⁺ binding sites, with aspartic acid residues (like the one at position 244) being particularly important for its function . CASQ1 has the ability to form elongated polymers within the sarcoplasmic reticulum, which is critical for its calcium-sequestering capability . Mutations in the CASQ1 gene can lead to vacuolar myopathies characterized by weakness, fatigue, and the formation of protein aggregates within muscle cells .

• What are the optimal applications for different types of CASQ1 antibodies?

CASQ1 antibodies can be effectively utilized in multiple research applications with varying optimal conditions:

• Immunoblotting/Western blotting: Polyclonal antibodies typically perform well at concentrations of 0.04-0.4 μg/mL . This technique is particularly useful for detecting CASQ1 in triad samples or muscle lysates, allowing for protein quantification and identification of potential mutations .

• Immunohistochemistry: Recommended dilutions range from 1:20 to 1:50 for optimal staining . This application is valuable for visualizing CASQ1 distribution in muscle biopsies and identifying pathological features such as vacuoles in muscle fibers .

• Immunofluorescence: Both monoclonal and polyclonal antibodies can be used effectively, with proper secondary antibody selection (e.g., Cy2-conjugated for goat primaries, Cy3-conjugated for mouse or rabbit primaries) . This technique provides high-resolution visualization of CASQ1 localization within cellular compartments.

• ELISA: Multiple antibody formats including unconjugated, HRP-conjugated, FITC-conjugated, and biotin-conjugated antibodies are available for this application .

• How can I validate the specificity of a CASQ1 antibody for my experimental system?

Validating CASQ1 antibody specificity requires a multi-faceted approach:

• Positive controls: Use skeletal muscle tissue samples known to express CASQ1 abundantly. Human, mouse, and rat tissues are commonly used, with antibodies showing cross-reactivity across these species .

• Recombinant protein validation: Test antibodies against recombinant CASQ1 protein expressed in systems like E. coli (using pGEX-4T-1 vector) or mammalian cell lines like COS-7 cells . This approach confirms direct binding to the target protein.

• Knockout/knockdown controls: When available, use CASQ1-knockout tissue or cells with siRNA-mediated CASQ1 knockdown as negative controls.

• Immunoprecipitation cross-validation: Perform co-immunoprecipitation assays using anti-CASQ1 antibodies and verify results with antibodies targeting known CASQ1-interacting proteins like STIM2 .

• Enhanced validation techniques: Consider orthogonal RNAseq validation or recombinant expression validation as recommended for Prestige Antibodies .

• What is the optimal sample preparation protocol for CASQ1 immunodetection in muscle tissues?

Sample preparation for CASQ1 immunodetection varies by experimental approach:

For immunohistochemistry and immunofluorescence:

• Fix fresh muscle tissue sections with 3% paraformaldehyde in PBS for optimal preservation of CASQ1 structure and localization .

• Permeabilize samples with HEPES-Triton Buffer to allow antibody access to intracellular CASQ1 .

• Block non-specific binding using a solution containing 0.5% BSA and 2% horse serum in PBS for 30 minutes .

• Incubate with primary antibodies at 4°C overnight, followed by appropriate secondary antibody incubation at room temperature for 1 hour .

• For fluorescence microscopy, mount slides with DAPI stain for nuclear visualization .

For protein extraction and immunoblotting:

• Prepare muscle lysates using specialized buffers compatible with membrane protein extraction.

• For triad vesicle preparation (enriched with CASQ1 and other calcium-handling proteins), follow established protocols for optimal purification .

• For coimmunoprecipitation assays, solubilize myotube lysate (approximately 100 μg total protein) before adding anti-CASQ1 antibodies .

• What are the key considerations when selecting antibodies for different CASQ1 protein domains?

When selecting domain-specific CASQ1 antibodies, consider these factors:

• Functional domains: Antibodies targeting different regions of CASQ1 (e.g., AA 29-130, AA 127-396, AA 136-381) provide different insights . The calcium-binding domains (containing aspartic acid residues like D244) are particularly relevant for functional studies .

• Species conservation: Choose antibodies targeting conserved regions if cross-species reactivity is needed. The immunogen sequence should be evaluated against the target species' CASQ1 sequence .

• Host species compatibility: Consider the host species (mouse, rabbit) in relation to your experimental design, especially for co-labeling studies to avoid cross-reactivity between secondary antibodies .

• Clonality considerations: Monoclonal antibodies offer high specificity for particular epitopes, while polyclonal antibodies may provide stronger signals by recognizing multiple epitopes .

• Tag recognition: If working with tagged recombinant CASQ1 constructs (such as GST-CASQ1), ensure the antibody's epitope is not masked by the tag .

Advanced Research Questions

• How can CASQ1 antibodies be optimized for studying pathogenic protein aggregation in CASQ1-related myopathies?

CASQ1-related myopathies involve specific pathological features including protein aggregation that require specialized antibody-based detection approaches:

• Serial section analysis: To properly characterize CASQ1 aggregates, implement serial section immunolabeling with antibodies against CASQ1 and other sarcoplasmic reticulum proteins. This allows tracking of individual muscle fibers across sections and correlation of vacuole formation with protein accumulation patterns .

• Double immunofluorescence labeling: Combine CASQ1 antibodies with markers for other sarcoplasmic reticulum proteins to identify co-aggregation patterns. In CASQ1 p.Asp244Gly mutations, vacuoles show strong immunoreactivity for sarcoplasmic reticulum proteins, while in p.Gly103Asp mutations, a different pattern with tubular aggregates is observed .

• Fiber type-specific analysis: Since CASQ1 mutations like p.Asp244Gly preferentially affect type II fibers, combine CASQ1 immunolabeling with fiber-type markers to distinguish pathology patterns between fiber types .

• Transmission electron microscopy with immunogold labeling: For ultrastructural characterization of electron-dense SR vacuoles containing mutant CASQ1 protein aggregates, immunogold labeling with CASQ1 antibodies can provide nanometer-scale resolution of aggregate composition .

• Confocal microscopy optimization: Use high-resolution confocal microscopy (e.g., ZEISS LSM 510) with appropriate secondary antibodies (Cy3 or Cy2-conjugated) to visualize the detailed morphology of CASQ1 aggregates .

• What methodological approaches can be used to investigate CASQ1 mutations and their effects on protein function?

Investigating CASQ1 mutations requires an integrated approach utilizing antibody-based techniques:

• Heterologous expression systems: Express wild-type and mutant CASQ1 (e.g., p.Asp244Gly, p.Gly103Asp) in cell models like COS-7 cells and analyze differences in protein behavior using immunofluorescence with anti-CASQ1 antibodies. This approach revealed that the p.Asp244Gly mutation results in markedly reduced ability to form elongated polymers .

• In vivo mouse fiber transduction: Combine viral-mediated gene transfer of mutant CASQ1 constructs with immunofluorescence analysis to study mutation effects in physiological contexts .

• Molecular dynamics simulation: Complement antibody studies with computational approaches like molecular dynamics simulations (e.g., 40 ns simulations using NAMD with CHARMM 27 force field) to predict how mutations affect protein structure and folding stability .

• Calcium kinetics analysis: Correlate immunolocalization of wild-type versus mutant CASQ1 with functional calcium release kinetics measurements to establish structure-function relationships .

• Phosphorylation site analysis: Use phospho-specific antibodies alongside regular CASQ1 antibodies to investigate if mutations affect post-translational modifications predicted by tools like PhospoSitePlus .

• What are the optimal experimental designs for using CASQ1 antibodies to study protein-protein interactions in the calcium release complex?

Studying CASQ1's interactions with other proteins requires specialized experimental designs:

• GST pull-down assays: Express GST-tagged CASQ1 or specific CASQ1 regions in E. coli using vectors like pGEX-4T-1 and immobilize on GST beads. Incubate with triad samples (enriched with potential interaction partners) followed by immunoblotting with antibodies against suspected interaction partners like STIM2 .

• Co-immunoprecipitation optimization: For muscle samples, solubilize approximately 100 μg of total protein from myotube lysate and immunoprecipitate with anti-CASQ1 antibody. Analyze precipitates by immunoblotting with antibodies against potential interaction partners (e.g., STIM2, Orai1) .

• Recombinant protein domain mapping: Generate constructs expressing different CASQ1 regions (using primers as described in search result ) to identify specific interaction domains through pull-down assays followed by immunoblotting .

• Proximity ligation assays: Combine primary antibodies against CASQ1 and potential interaction partners to visualize protein-protein interactions in situ with nanometer resolution.

• FRET-based interaction studies: Use fluorescently-tagged antibodies against CASQ1 and interacting proteins to measure Förster resonance energy transfer as evidence of molecular proximity.

• How can researchers optimize detection of CASQ1 expression in different muscle fiber types and its alterations in myopathies?

Optimizing CASQ1 detection across different fiber types and pathological conditions requires:

• Fiber type correlation: Track single fibers through serial sections using CASQ1 antibodies in combination with fiber-type specific markers. Count and classify fibers according to type, presence of vacuoles (single or multiple), and immunoreactivity patterns .

• Image processing optimization: After acquisition using appropriate microscopy systems (e.g., Olympus BX60 equipped with DP70 charge-coupled device camera), process images with software like ImageJ to enhance visualization of CASQ1 distribution patterns .

• Quantitative immunofluorescence: Implement standardized image acquisition and analysis protocols to quantify CASQ1 immunofluorescence intensity across different fiber types and in pathological versus normal tissues.

• Correlation with clinical parameters: In CASQ1-related myopathies, correlate immunohistochemical findings with clinical data including age of onset, CK levels (which can range from 1-75 times normal), and specific symptoms such as exercise intolerance and myalgia .

• Muscle MRI correlation: Integrate CASQ1 immunohistochemistry findings with muscle MRI patterns, which show characteristic fibrofatty substitution patterns in CASQ1-related myopathies .

• What technical approaches can resolve contradictory findings when using different CASQ1 antibodies in research?

When faced with contradictory results from different CASQ1 antibodies, implement these technical approaches:

• Epitope mapping: Determine the exact epitopes recognized by different antibodies using peptide arrays or deletion constructs. Antibodies targeting different regions (e.g., AA 29-130 versus AA 127-396) may yield different results depending on protein conformation or masking in complexes .

• Cross-validation with multiple antibodies: Validate findings using at least two different antibodies targeting distinct CASQ1 epitopes, preferably from different host species and with different clonality (monoclonal versus polyclonal) .

• Genetic validation: Corroborate antibody findings with genetic approaches such as CASQ1 gene sequencing, especially when investigating potential pathogenic mutations like p.Asp244Gly or p.Gly103Asp .

• Western blot analysis with denaturing versus non-denaturing conditions: Compare results under different conditions to determine if contradictions arise from conformational differences affecting epitope accessibility.

• Mass spectrometry validation: Use antibody-independent proteomic approaches to confirm CASQ1 presence, abundance, and modifications when antibody results conflict.

• RNA expression correlation: Correlate protein detection results with mRNA expression data to help resolve contradictory immunodetection findings.