HRC Antibody

What is HRC and why is it an important research target?

HRC (histidine-rich calcium binding protein) is a protein with 699 amino acid residues and a mass of 80.2 kDa in humans, primarily expressed in testis, skeletal muscle, and heart muscle. It plays a critical role in regulating calcium sequestration or release in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle . HRC has gained research importance due to its involvement in various physiological processes and pathological conditions, including cardiac function regulation and cancer progression . Studies have shown that HRC is frequently upregulated in hepatocellular carcinoma (HCC), where it promotes tumor progression and metastasis, making it a significant target for both basic research and potential therapeutic interventions .

What applications are HRC antibodies commonly used for?

HRC antibodies are widely employed in multiple research applications:

The selection of application depends on the specific research question being addressed. For instance, Western blotting is commonly used for expression level studies, while IHC provides insights into tissue distribution patterns of HRC .

How should HRC antibodies be stored and handled for optimal performance?

For maximum antibody stability and performance, HRC antibodies should generally be stored at -20°C. Most commercial HRC antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Long-term stability is typically guaranteed for one year after shipment when properly stored. While some manufacturers suggest aliquoting is unnecessary for -20°C storage, it is generally good practice to minimize freeze-thaw cycles. When working with HRC antibodies, it's important to use appropriate controls and follow manufacturer-recommended protocols for each specific application to ensure optimal results .

What are typical positive controls for validating HRC antibody specificity?

Validated positive controls for HRC antibodies include:

• Mouse heart tissue

• Mouse skeletal muscle tissue

• HeLa cells

These tissues/cells have confirmed HRC expression and serve as reliable positive controls for antibody validation. When establishing a new experimental system, researchers should include these controls alongside experimental samples to confirm antibody specificity and performance .

How should I design an optimal antibody titration for HRC detection in Western blot applications?

When titrating HRC antibodies for Western blot, follow this methodological approach:

• Begin with a dilution range based on manufacturer recommendations (typically 1:500-1:2000 for HRC antibodies)

• Prepare a gradient of at least 4-5 different dilutions (e.g., 1:500, 1:1000, 1:1500, 1:2000)

• Use a consistent amount of protein from a positive control sample (e.g., mouse heart tissue or skeletal muscle)

• Process all blots under identical conditions (transfer time, blocking, washing)

• Compare signal-to-background ratio across different dilutions

• Select the optimal dilution that provides maximum specific signal with minimal background

• Validate the chosen dilution with experimental samples

This systematic approach ensures reproducible and reliable detection of HRC in your experimental system while minimizing antibody consumption .

How can I troubleshoot non-specific binding issues when using HRC antibodies in immunohistochemistry?

Non-specific binding in IHC with HRC antibodies can be systematically addressed through:

• Optimize antigen retrieval: HRC antibodies typically perform best with TE buffer pH 9.0, though citrate buffer pH 6.0 may be used as an alternative

• Adjust blocking conditions: Increase blocking time (1-2 hours) and consider testing different blocking agents (5% BSA, 5-10% normal serum, commercial blockers)

• Titrate primary antibody: Test multiple dilutions (starting with 1:50-1:500 range)

• Increase washing steps: Add additional washing steps with 0.1-0.3% Tween-20 in PBS

• Use proper controls:

  • Negative controls (omit primary antibody)

  • Positive controls (mouse heart tissue)

  • Isotype controls (irrelevant antibody of same isotype)

• Consider tissue-specific autofluorescence: Employ autofluorescence quenching reagents if needed

• Test different detection systems: HRP/DAB vs. fluorescent secondary antibodies

Systematic documentation of each modification will help identify the optimal conditions for specific HRC detection in your tissue samples .

What is the best approach for incorporating HRC antibodies into multi-parameter flow cytometry panels?

When incorporating HRC antibodies into multi-parameter flow cytometry panels, follow this systematic approach:

• Determine compatibility with surface/intracellular staining protocols: HRC is primarily intracellular, requiring appropriate permeabilization

• Select appropriate fluorophore conjugate: Choose based on:

  • Instrument configuration and available detectors

  • Abundance of HRC in target population (bright fluorophores for low-abundance targets)

  • Potential spectral overlap with other markers

• Perform antibody titration: Establish optimal signal-to-noise ratio

• Create proper compensation controls: Single-stained controls for each fluorophore

• Evaluate spectral unmixing: Critical for full-spectrum flow cytometry applications

• Validate with FMO controls: Fluorescence Minus One controls help identify true positive populations

• Manage autofluorescence: Implement autofluorescence subtraction strategies if needed

For full-spectrum flow cytometry applications, additional considerations include:

• Preparation of optimal spectral reference controls

• Unmixing evaluation of fully stained samples

• Management of heterogeneous autofluorescence patterns as outlined in current protocols

How can computational models be leveraged to predict and improve HRC antibody specificity?

Advanced computational approaches can significantly enhance HRC antibody specificity through:

• Biophysics-informed modeling: Implementing models that associate distinct binding modes with potential ligands to predict specific variants beyond experimentally observed sequences

• Phage display integration: Combining computational predictions with phage display experiments where antibodies are selected against diverse combinations of ligands

• Energy function optimization: Customizing antibody sequences by optimizing energy functions (E_sw) associated with each binding mode:

  • For cross-specific antibodies: Jointly minimize functions associated with desired ligands

  • For specific antibodies: Minimize functions for desired ligands while maximizing those for undesired ligands

• Machine learning applications: Leveraging high-throughput sequencing data and machine learning to make predictions beyond experimentally observed sequences

• Counter-selection strategies: Implementing computational counter-selection to eliminate off-target binding more efficiently than experimental approaches

These computational approaches can generate novel antibody variants with customized specificity profiles, either allowing interaction with several distinct ligands (cross-specific) or enabling interaction with a single ligand while excluding others (specific) .

What molecular mechanisms explain the discrepancy between predicted (80 kDa) and observed (140-150 kDa) molecular weights of HRC in Western blot analysis?

The discrepancy between the calculated molecular weight of HRC (80 kDa) and its observed molecular weight (140-150 kDa) in Western blot analysis can be explained by several molecular mechanisms:

• Post-translational modifications:

  • Extensive glycosylation of HRC

  • Phosphorylation at multiple sites

  • Other modifications affecting protein migration

• Structural features:

  • The histidine-rich regions of HRC may bind SDS inefficiently

  • Unusual amino acid composition affecting electrophoretic mobility

  • Tertiary structure resistance to complete denaturation

• Methodological considerations:

  • Running conditions (buffer pH, temperature)

  • Gel percentage selection

  • Sample preparation methods

To accurately interpret Western blot results, researchers should:

• Include appropriate molecular weight markers

• Consider alternative confirmation methods (mass spectrometry)

• Validate with recombinant protein controls of known molecular weight

This phenomenon is not unique to HRC and has been observed with other proteins containing unusual amino acid compositions or extensive post-translational modifications.

How might HRC antibodies be utilized to investigate the role of HRC in hepatocellular carcinoma progression and metastasis?

HRC antibodies can be strategically employed to investigate HRC's role in hepatocellular carcinoma (HCC) through several sophisticated approaches:

• Comprehensive tissue expression profiling:

  • Tissue microarray analysis of HCC specimens at different stages

  • Correlation of HRC expression with clinical parameters (tumor size, metastasis)

  • Multi-parameter IHC to examine co-expression with other markers (SATB1)

• Mechanistic studies in cellular models:

  • Immunofluorescence for subcellular localization in HCC cells

  • Co-immunoprecipitation to identify HRC-interacting proteins

  • Chromatin immunoprecipitation to study SATB1-mediated HRC regulation

• In vivo models:

  • IHC analysis of orthotopic xenograft models comparing HRC-overexpressing vs. control cells

  • Correlation of HRC expression with metastatic potential using bioluminescent imaging

• Therapeutic approach development:

  • Screen for compounds that modulate HRC expression/function

  • Develop HRC-targeting antibodies for potential therapeutic applications

  • Evaluate effects of SATB1 inhibitors on HRC expression and HCC progression

Evidence suggests HRC promotes HCC metastasis through pathways that may involve focal adhesion turnover and is potentially regulated by SATB1, which activates HRC gene transcription primarily through JNK-dependent AP-1 activation .

What criteria should be used to validate the specificity of a new commercial HRC antibody?

Rigorous validation of new commercial HRC antibodies should include:

• Multiple application testing:

  • Western blot detection of expected 140-150 kDa band in positive control tissues

  • IHC demonstration of expected tissue distribution patterns

  • IF/ICC showing correct subcellular localization

• Positive control validation:

  • Detection in known HRC-expressing tissues (heart, skeletal muscle)

  • Consistent results across multiple cell lines with known HRC expression

• Specificity controls:

  • Preabsorption with immunizing peptide should abolish signal

  • Testing in HRC knockout/knockdown models should show reduced/absent signal

  • Comparison with alternative HRC antibodies targeting different epitopes

• Cross-reactivity assessment:

  • Testing across multiple species if claimed (human, mouse, etc.)

  • Evaluation in tissues known to lack HRC expression

• Reproducibility testing:

  • Lot-to-lot consistency assessment

  • Inter-laboratory validation when possible

How can I develop a quantitative ELISA assay for measuring HRC levels in cardiac tissue samples?

Developing a quantitative ELISA for HRC measurement in cardiac tissues requires systematic optimization:

• Antibody pair selection:

  • Test multiple capture/detection antibody combinations targeting different HRC epitopes

  • Validate each pair for specificity using recombinant HRC protein

  • Ensure antibodies recognize native HRC conformation

• Sample preparation optimization:

  • Develop standardized tissue homogenization protocol

  • Optimize protein extraction buffer (consider detergent types/concentrations)

  • Establish protein concentration normalization method

• Assay development:

  • Determine optimal antibody concentrations through checkerboard titration

  • Establish blocking conditions minimizing background

  • Optimize incubation times and temperatures

• Standard curve generation:

  • Use purified recombinant HRC protein for calibration

  • Prepare standards in identical matrix as samples

  • Validate linear range and detection limits

• Validation parameters:

  • Intra-assay coefficient of variation (<10%)

  • Inter-assay coefficient of variation (<15%)

  • Spike-and-recovery experiments (80-120% recovery)

  • Dilutional linearity assessment

  • Sample stability testing

• Clinical validation:

  • Compare results with established methods (Western blot)

  • Analyze samples from normal and diseased cardiac tissues

Documentation of each optimization step ensures reproducibility and reliability of the final quantitative ELISA protocol .

How can HRC antibodies be utilized to investigate calcium handling dysregulation in cardiac pathologies?

HRC antibodies can effectively investigate calcium handling dysregulation in cardiac pathologies through:

• Expression level analysis:

  • Western blot quantification of HRC in diseased vs. healthy cardiac tissues

  • IHC assessment of expression patterns in different cardiomyopathies

  • Correlation of HRC levels with disease progression markers

• Subcellular localization studies:

  • High-resolution immunofluorescence to track HRC redistribution in diseased hearts

  • Co-localization with other calcium handling proteins (SERCA2, RyR2)

  • Super-resolution microscopy to examine nanodomain organization changes

• Protein-protein interaction investigation:

  • Co-immunoprecipitation to identify altered HRC interactions in disease states

  • Proximity ligation assays to visualize HRC interactions in situ

  • Pull-down assays to study calcium-dependent binding partners

• Post-translational modification assessment:

  • Phosphorylation-specific antibodies to study HRC regulation

  • Analysis of changes in glycosylation patterns

  • Correlation of modifications with functional outcomes

• Intervention studies:

  • Monitoring HRC expression/localization following therapeutic interventions

  • Analysis of HRC in heart failure progression and recovery

  • Correlation of HRC changes with functional cardiac parameters

HRC has been implicated in calcium sequestration or release in the sarcoplasmic reticulum, making it a valuable target for understanding mechanisms of cardiac dysfunction where calcium handling is disrupted .

What methods can be used to investigate the regulatory relationship between SATB1 and HRC in cancer models?

To investigate the regulatory relationship between SATB1 and HRC in cancer models, researchers can employ these methodological approaches:

• Expression correlation analysis:

  • Quantitative RT-PCR to measure mRNA levels of both genes across clinical samples

  • Western blot to assess protein expression correlation

  • Statistical analysis of Pearson's correlation coefficient (reported as 0.494 in HCC tissues)

• Genetic manipulation studies:

  • SATB1 knockdown (siRNA/shRNA) to observe effects on HRC expression

  • SATB1 overexpression to confirm upregulation of HRC

  • Rescue experiments combining SATB1 knockdown with HRC overexpression

• Promoter activity assessment:

  • Luciferase reporter assays with HRC promoter constructs

  • ChIP assays to detect SATB1 binding to HRC promoter regions

  • EMSA to confirm direct DNA-protein interactions

• Signaling pathway investigation:

  • Inhibitor studies targeting JNK pathway components

  • Phosphorylation analysis of AP-1 transcription factors

  • Time-course experiments to establish causality

• In vivo validation:

  • Xenograft models with SATB1 manipulation

  • Correlation of SATB1/HRC expression in tumor tissues

  • Therapeutic targeting of this regulatory axis

Evidence suggests SATB1 enhances HRC gene transcription by activating AP-1 primarily through JNK-dependent mechanisms. Among tissues with elevated SATB1, approximately 76.9% also expressed high levels of HRC, supporting this regulatory relationship .

How might new computational approaches enhance the design of highly specific HRC antibodies for research and therapeutic applications?

Emerging computational approaches offer significant potential for designing highly specific HRC antibodies:

• AI-driven epitope mapping:

  • Deep learning algorithms to identify unique HRC epitopes

  • Molecular dynamics simulations to evaluate epitope accessibility

  • Structure-based design of complementary binding regions

• Biophysics-informed modeling frameworks:

  • Implementation of models associating distinct binding modes with specific ligands

  • Optimization of binding specificity through energy function analysis

  • Generation of novel antibody sequences with predetermined binding profiles

• Integrated experimental-computational pipelines:

  • Phage display experiments coupled with high-throughput sequencing

  • Machine learning analysis of selection outcomes

  • Iterative refinement of computational predictions

• Specificity profile customization:

  • Design of cross-specific antibodies by jointly minimizing energy functions

  • Creation of highly specific antibodies by minimizing desired interactions while maximizing unwanted ones

  • Generation of antibodies targeting specific HRC conformations

• Developability assessment integration:

  • Early evaluation of antibody properties like solubility and stability

  • Prediction of immunogenicity risks

  • Optimization for manufacturability alongside specificity

These approaches can accelerate the development of HRC antibodies with precisely defined specificity profiles, potentially enabling both improved research tools and therapeutic candidates with reduced off-target effects .

What potential exists for using HRC antibodies in the development of targeted therapies for hepatocellular carcinoma?

The development of HRC-targeted therapies for hepatocellular carcinoma shows promising potential based on current research findings:

• Therapeutic antibody development:

  • Generation of humanized antibodies targeting extracellular or exposed HRC epitopes

  • Development of antibody-drug conjugates delivering cytotoxic payloads

  • Bi-specific antibodies linking HRC-expressing cells to immune effectors

• Mechanistic intervention strategies:

  • Targeting the HRC-mediated promotion of tumor metastasis

  • Interfering with focal adhesion turnover pathways

  • Disrupting the SATB1-HRC regulatory axis

• Diagnostic and therapeutic integration:

  • HRC antibodies for patient stratification (correlating with tumor size, metastasis)

  • Monitoring treatment response through HRC expression

  • Combination approaches targeting multiple metastasis-promoting factors

• Delivery system development:

  • Nanoparticle-conjugated antibodies for enhanced tumor penetration

  • Liver-targeted delivery systems to increase specificity

  • Stimuli-responsive release mechanisms for precise targeting

• Translational considerations:

  • Biomarker development correlating HRC expression with clinical outcomes

  • Patient selection strategies based on HRC/SATB1 expression profiles

  • Combination with existing HCC standard-of-care treatments

With HRC significantly upregulated in 67.47% of HCC specimens and its expression correlating with tumor size (P=0.026) and metastasis (P=0.004), it represents a promising therapeutic target. In vivo studies have already demonstrated that HRC overexpression enhances intrahepatic and lung metastasis in orthotopic xenograft models, further supporting its potential as a therapeutic target .

How can I optimize immunofluorescence protocols for simultaneous detection of HRC and other calcium-handling proteins?

Optimizing multiplex immunofluorescence for HRC and other calcium-handling proteins requires:

• Antibody compatibility assessment:

  • Select HRC antibodies from different host species than other target antibodies

  • Verify each antibody individually before multiplexing

  • Test for cross-reactivity between secondary antibodies

• Sample preparation optimization:

  • Determine optimal fixation method (4% PFA typically works well)

  • Optimize antigen retrieval conditions (TE buffer pH 9.0 recommended for HRC)

  • Evaluate different permeabilization protocols (0.1-0.3% Triton X-100)

• Sequential staining strategy:

  • Begin with lowest abundance target and most robust antibody

  • Apply blocking steps between sequential antibody applications

  • Consider tyramide signal amplification for weak signals

• Fluorophore selection:

  • Choose spectrally distinct fluorophores to minimize bleed-through

  • Account for tissue autofluorescence spectrum in channel selection

  • Match fluorophore brightness to target abundance

• Imaging optimization:

  • Acquire single-stained controls for spectral unmixing

  • Implement appropriate background subtraction methods

  • Use sequential scanning when possible to minimize crosstalk

• Validation controls:

  • Include absorption controls to verify specificity

  • Use tissue with known expression patterns of all targets

  • Quantify colocalization with appropriate statistical measures

For HRC specifically, a dilution range of 1:50-1:500 is typically recommended for immunofluorescence applications .

What considerations are important when selecting an HRC antibody for cross-species studies?

When selecting HRC antibodies for cross-species studies, consider these critical factors:

• Epitope conservation analysis:

  • Perform sequence alignment of HRC across target species

  • Identify regions of high conservation for antibody selection

  • Verify epitope presence in species of interest (human HRC vs. mouse, rat, bovine, etc.)

• Validated cross-reactivity:

  • Select antibodies explicitly tested across multiple species

  • Review validation data from manufacturers showing species reactivity

  • Consider antibodies raised against synthetic peptides matching conserved regions

• Application-specific validation:

  • Test antibody in each application (WB, IHC, IF) for each species

  • Verify expected molecular weight differences between species

  • Assess potential background differences across species

• Positive control selection:

  • Identify appropriate positive control tissues for each species

  • Heart and skeletal muscle tissues are suitable for most species

  • Include human samples as reference when studying human orthologs

• Protocol optimization:

  • Adjust dilutions for different species (may require higher concentration)

  • Modify antigen retrieval conditions for different tissues

  • Adapt blocking protocols to minimize species-specific background

Many commercial HRC antibodies show reactivity with human, mouse, rabbit, rat, bovine, dog, goat, guinea pig, horse, and yeast samples, though validation extent varies by manufacturer and should be critically evaluated .

What strategies can resolve inconsistent HRC detection in Western blot applications?

Resolving inconsistent HRC detection in Western blots requires systematic troubleshooting:

• Sample preparation optimization:

  • Improve protein extraction with specialized buffers containing protease inhibitors

  • Test different lysis conditions (RIPA vs. NP-40 vs. Triton X-100)

  • Standardize protein quantification and loading (30-50μg recommended)

• Gel electrophoresis modifications:

  • Adjust acrylamide percentage (8% gels typically work well for 140-150 kDa HRC)

  • Optimize running conditions (voltage, time, temperature)

  • Consider gradient gels for better resolution of high molecular weight proteins

• Transfer parameters adjustment:

  • Test different transfer methods (wet vs. semi-dry)

  • Increase transfer time for high molecular weight HRC (140-150 kDa)

  • Add SDS to transfer buffer to improve large protein transfer

• Antibody optimization:

  • Titrate primary antibody (1:500-1:2000 range recommended)

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

  • Test different antibody clones targeting different HRC epitopes

• Detection system enhancement:

  • Switch between ECL, advanced ECL, or fluorescent detection

  • Optimize exposure times (HRC may require longer exposures)

  • Consider signal amplification systems for low abundance samples

• Positive control inclusion:

  • Always run mouse heart or skeletal muscle tissue as positive control

  • Include recombinant HRC protein when available

  • Compare results with published literature for expected band patterns

By systematically addressing these parameters, researchers can achieve consistent and specific detection of HRC in Western blot applications .

How can background reduction be achieved when using HRC antibodies in immunohistochemistry of tissues with high endogenous biotin?

For reducing background in IHC with HRC antibodies in biotin-rich tissues:

• Biotin blocking system implementation:

  • Apply avidin solution (15 minutes)

  • Wash thoroughly

  • Apply biotin solution (15 minutes)

  • Complete this before primary antibody incubation

• Alternative detection system selection:

  • Use non-biotin polymer detection systems

  • Consider HRP-conjugated secondary antibodies

  • Employ fluorescent secondary antibodies instead of biotin-based detection

• Endogenous enzyme blocking enhancement:

  • Extend peroxidase blocking (3% H₂O₂, 10-15 minutes)

  • Add additional blocking steps (levamisole for alkaline phosphatase)

  • Consider dual enzyme block solutions

• Background reduction techniques:

  • Increase blocking serum concentration (5-10%)

  • Add protein blockers (1-2% BSA, casein, or commercial blockers)

  • Include 0.1-0.3% Triton X-100 in antibody diluent

  • Apply longer blocking periods (1-2 hours)

• Antibody optimization:

  • Further dilute primary antibody (start with 1:500)

  • Reduce incubation temperature (4°C overnight)

  • Add normal serum from secondary antibody host species to diluent

• Tissue preparation considerations:

  • Optimize fixation time (over-fixation increases background)

  • Test different antigen retrieval methods (TE buffer pH 9.0 recommended)

  • Evaluate fresh frozen vs. FFPE sections for your specific application