CASQ2 Antibody

What are the optimal applications for CASQ2 antibody detection?

CASQ2 antibody (such as 18422-1-AP) can be effectively used in multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), and ELISA. Each application requires specific optimization for reliable results. The antibody shows consistent reactivity with human, mouse, rat, and pig samples, making it versatile for comparative studies across species .

For optimal results, recommended dilutions are:

• Western Blot: 1:2000-1:16000

• Immunohistochemistry: 1:50-1:500

• Immunofluorescence/ICC: 1:50-1:500

It is crucial to note that optimal dilution is sample-dependent, and researchers should validate the antibody in their specific experimental system before proceeding with large-scale experiments.

How should CASQ2 antibody storage and handling be optimized for research applications?

For maximum stability and performance, CASQ2 antibodies should be stored at -20°C, where they typically remain stable for one year after shipment. The storage buffer generally consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is not necessary for -20°C storage, which simplifies lab management protocols.

When handling the antibody, minimize freeze-thaw cycles and maintain sterile conditions to preserve activity. Some preparations (20μl sizes) contain 0.1% BSA, which acts as a stabilizing agent during storage . Always centrifuge the antibody vial briefly before opening to ensure all liquid is at the bottom of the tube and follow manufacturer's instructions for specific handling requirements.

What is the expected molecular weight pattern for CASQ2 in Western blot applications?

When using CASQ2 antibodies in Western blot applications, researchers should expect to observe a band at approximately 50 kDa, which is the experimentally observed molecular weight for the protein. This differs slightly from the calculated molecular weight of 46 kDa based on amino acid sequence . This discrepancy is common for many proteins due to post-translational modifications or structural characteristics.

In knockout validation studies, researchers have confirmed CASQ2 antibody specificity by demonstrating absence of this band in CASQ2-null mouse models. For proper identification, positive controls using heart tissue from wild-type specimens are recommended, as cardiac tissue consistently shows strong CASQ2 expression .

How can researchers effectively design experiments to study CASQ2 expression in disease models associated with cardiac arrhythmias?

When designing experiments to study CASQ2 in cardiac arrhythmia models, researchers should implement a comprehensive approach that combines protein expression analysis with functional studies. CASQ2 mutations have been associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and appropriate experimental design should account for both basal conditions and stress responses .

For effective experimental design:

• Include appropriate controls:

  • Wild-type specimens

  • Heterozygous models (CASQ²⁺/⁻)

  • Homozygous knockout models (CASQ²⁻/⁻)

• Implement both basal and stress conditions:

  • Normal physiological conditions

  • Catecholamine exposure to induce stress responses

  • Exercise or electrical stimulation protocols

• Combine multiple detection methods:

  • Immunoblotting for protein quantification

  • Immunohistochemistry for localization studies

  • RNA analysis using real-time PCR

Research has shown that despite absent CASQ2, knockout models maintain relatively normal Ca²⁺ release and contractile function under basal conditions but show significant abnormalities under stress, suggesting compensatory mechanisms are involved .

What methodological approaches should be used to analyze compensatory changes in calcium handling proteins when studying CASQ2 mutations?

When studying CASQ2 mutations, it is critical to analyze compensatory changes in other calcium handling proteins, as these adaptations may significantly impact phenotypic expression. Research on CASQ2-null mice has revealed important compensatory mechanisms including increases in SR volume, reductions in triadin-1 and junctin levels, and upregulation of other Ca²⁺-binding proteins like calreticulin (CRT) .

Methodological approaches should include:

• Comprehensive protein analysis:

  • SDS-PAGE followed by immunoblotting for specific calcium handling proteins

  • Use ¹²⁵I–protein A for quantification with phosphorimaging

  • ⁴⁵Ca²⁺ overlay techniques to detect multiple Ca²⁺-binding proteins in SR vesicles

• Comparative RNA expression analysis:

  • Real-time PCR using appropriate primers for calcium handling proteins

  • Standardization against housekeeping genes like β-microtubulin

  • RACE (Rapid Amplification of cDNA Ends) techniques for full transcript analysis

• Structural analysis:

  • Electron microscopy to assess SR volume changes

  • Immunolocalization studies to evaluate protein distribution changes

These methodological approaches allow for comprehensive assessment of the molecular adaptations that occur in response to CASQ2 deficiency .

How can CASQ2 antibodies be utilized to investigate the structural and functional relationships between cardiac calsequestrin and associated proteins?

CASQ2 antibodies are valuable tools for investigating the structural and functional relationships between cardiac calsequestrin and its associated proteins, particularly in the context of calcium release complexes in the sarcoplasmic reticulum. A comprehensive experimental approach should incorporate multiple techniques:

• Co-immunoprecipitation studies:

  • Use CASQ2 antibodies to pull down protein complexes

  • Analyze associated proteins such as triadin-1, junctin, and RyR2

  • Compare complex formation in normal versus pathological conditions

• Proximity ligation assays:

  • Combine CASQ2 antibodies with antibodies against potential interaction partners

  • Visualize and quantify protein-protein interactions in situ

  • Assess how mutations affect these interactions

• Immunofluorescence co-localization:

  • Apply CASQ2 antibodies (1:50-1:500 dilution) alongside antibodies for other SR proteins

  • Use confocal microscopy to assess spatial relationships

  • Quantify co-localization using appropriate statistical methods

Research has demonstrated that CASQ2 functions as part of a macromolecular complex including triadin-1 and junctin, which modulates RyR2 function. When CASQ2 is absent, significant reductions in triadin-1 and junctin levels occur, suggesting interconnected regulation . These studies help elucidate how alterations in one component of the calcium-handling machinery can lead to compensatory changes in others.

What are the common issues encountered when using CASQ2 antibodies in immunohistochemistry, and how can they be resolved?

Researchers frequently encounter challenges when using CASQ2 antibodies for immunohistochemistry. These issues and their solutions include:

• Weak or absent signal:

  • Try different antigen retrieval methods: The recommended approach is TE buffer pH 9.0, but citrate buffer pH 6.0 can be used as an alternative

  • Optimize antibody concentration: Starting with 1:50 dilution and titrating to 1:500 based on signal strength

  • Increase incubation time or temperature

  • Ensure tissue preservation method is compatible with the epitope

• High background staining:

  • Implement additional blocking steps with BSA or serum

  • Reduce antibody concentration

  • Include additional washing steps

  • Use tissue from CASQ2-knockout models as negative controls to confirm specificity

• Inconsistent staining patterns:

  • Standardize fixation protocols

  • Control incubation conditions precisely

  • Process all experimental samples simultaneously

  • Use positive control tissues known to express CASQ2 (heart tissue is ideal)

For optimal results in human tissues, CASQ2 antibodies have shown positive IHC detection in heart, kidney, ovary, placenta, skin, and spleen tissues . When analyzing results, consider that CASQ2 expression patterns may vary across different cardiac regions and may change under pathological conditions.

How should researchers address discrepancies between CASQ2 protein detection and functional data in experimental models?

When facing discrepancies between CASQ2 protein detection and functional data, researchers should implement a systematic troubleshooting approach:

• Validate antibody specificity:

  • Confirm antibody specificity using CASQ2-null models as negative controls

  • Verify results with multiple antibodies targeting different epitopes of CASQ2

  • Consider using a monoclonal antibody that recognizes downstream residues (e.g., residues 264-272) for confirmation

• Assess post-translational modifications:

  • The observed molecular weight (50 kDa) differs from the calculated weight (46 kDa), suggesting modifications

  • Implement phosphorylation-specific detection methods

  • Use Stains-all staining to detect Ca²⁺-binding proteins in SR vesicles

• Evaluate compensatory mechanisms:

  • Measure expression of other calcium-handling proteins that may compensate for CASQ2 deficiency

  • Assess structural changes in the SR that might maintain calcium storage capacity

  • Analyze functional parameters under both basal and stress conditions

Research has shown that CASQ2-null mice maintain relatively normal cardiac contractility and calcium handling under basal conditions despite the absence of CASQ2 protein. This apparent contradiction is resolved by discovering significant compensatory changes, including increased SR volume and alterations in other calcium-handling proteins, which become inadequate only under stress conditions .

What quality control measures should be implemented when using CASQ2 antibodies in multi-species comparative studies?

When conducting comparative studies across species using CASQ2 antibodies, rigorous quality control measures are essential to ensure reliable and comparable results:

• Cross-reactivity validation:

  • Confirm antibody reactivity with each species being studied

  • The 18422-1-AP CASQ2 antibody shows documented reactivity with human, mouse, rat, and pig samples

  • Perform Western blot validation for each species before proceeding with other applications

• Epitope conservation analysis:

  • Analyze sequence homology of the immunogen region across species

  • The immunogen for many CASQ2 antibodies is a fusion protein (Ag13246)

  • Consider epitope-specific antibodies when sequence divergence exists

• Standardized protocols across species:

  • Adjust tissue processing based on species-specific requirements

  • Maintain consistent antibody dilutions (WB: 1:2000-1:16000; IHC: 1:50-1:500; IF: 1:50-1:500)

  • Implement identical detection systems and quantification methods

• Species-specific controls:

  • Include positive controls from each species

  • Where available, include knockout or knockdown samples as negative controls

  • Use purified Casq2 protein as a standard when performing quantitative analyses

Properly controlled multi-species studies can provide valuable insights into evolutionary conservation of CASQ2 function and species-specific adaptations in calcium handling mechanisms.

How do different CASQ2 antibodies compare in their ability to detect various mutant forms associated with cardiac arrhythmias?

Different CASQ2 antibodies vary in their capacity to detect various mutant forms of the protein associated with cardiac arrhythmias. This variability depends on the epitope recognized by each antibody and how specific mutations affect protein structure:

• Epitope-specific considerations:

  • N-terminal targeting antibodies may fail to detect truncation mutations

  • C-terminal targeting antibodies would miss N-terminal frameshift mutations

  • For comprehensive analysis, use antibodies targeting different regions of CASQ2

• Detection capabilities for specific mutations:

  • Antibodies recognizing residues 264-272 successfully detect protein in models with exon 1 deletion but fail with exon 9 deletion

  • Polyclonal antibodies raised against full-length CASQ2 typically detect most point mutations but may show reduced signal intensity

  • Monoclonal antibodies offer higher specificity but might miss conformational variants

• Comparative performance across methods:

  • Western blot typically offers highest sensitivity for detecting mutant forms

  • Immunohistochemistry provides valuable localization data but may have lower sensitivity

  • Immunofluorescence allows for co-localization studies with other SR proteins

Research on CASQ2 mutation models has shown that while homozygous exon 9 deletion (CASQ ΔE9/ΔE9) mice completely lack detectable CASQ2 protein, other mutations may result in reduced protein levels rather than complete absence . This highlights the importance of selecting appropriate antibodies based on the specific mutations being studied.

What methodological approaches provide the most reliable quantification of CASQ2 protein expression changes in disease models?

For reliable quantification of CASQ2 protein expression changes in disease models, researchers should employ multiple complementary approaches:

• Western blot quantification:

  • Use ¹²⁵I–protein A for precise quantification with phosphorimaging

  • Include loading controls and standardization proteins

  • Implement dilution series to ensure measurements within linear range

  • Recommended antibody dilutions: 1:2000-1:16000

• Mass spectrometry-based proteomics:

  • Provides absolute quantification of CASQ2 and related proteins

  • Allows simultaneous measurement of multiple calcium-handling proteins

  • Identifies post-translational modifications that may affect function

• RNA and protein correlation:

  • Combine protein quantification with RT-qPCR analysis

  • Use primers targeting regions spanning multiple exons (e.g., exons 4-8)

  • Standardize against stable reference genes like β-microtubulin

• Immunohistochemical quantification:

  • Enables tissue-specific and subcellular localization analysis

  • Implement digital image analysis for quantitative assessment

  • Use antigen retrieval with TE buffer pH 9.0 for optimal detection

Studies comparing different CASQ2 mutations have demonstrated that protein levels do not always correlate with gene expression, highlighting the importance of multi-method approaches. For instance, while some mutations lead to complete absence of protein, others result in significant reduction despite detectable mRNA, suggesting post-transcriptional regulation or protein stability issues .

How should researchers integrate CASQ2 antibody-based analyses with functional calcium handling studies to comprehensively assess cardiac physiology?

Comprehensive assessment of cardiac physiology requires thoughtful integration of CASQ2 antibody-based analyses with functional calcium handling studies:

• Correlative structural-functional analysis:

  • Map CASQ2 expression patterns using immunohistochemistry (1:50-1:500 dilution)

  • Combine with calcium imaging in the same or adjacent tissue sections

  • Correlate CASQ2 distribution with local calcium release events

• Multi-parameter assessment protocol:

  • Quantify CASQ2 and related protein levels (triadin-1, junctin, RyR2) by immunoblotting

  • Measure SR calcium content and release kinetics

  • Assess compensatory structural changes like SR volume increases

  • Evaluate cardiac function under both basal and stress conditions

• Experimental design considerations:

  • Include both normal and pathological conditions

  • Implement catecholamine challenges to reveal hidden abnormalities

  • Compare responses in heterozygous versus homozygous models

• Integrated data analysis framework:

  • Correlate protein expression levels with functional parameters

  • Develop mathematical models to predict functional outcomes based on molecular changes

  • Consider compensatory mechanisms when interpreting results