HRH3 Recombinant Monoclonal Antibody

What is HRH3 and what biological functions does it serve?

HRH3 (Histamine H3 Receptor, also known as HH3R or GPCR97) belongs to the family 1 of G protein-coupled receptors. The human version has a canonical amino acid length of 445 residues and a protein mass of 48.7 kilodaltons, with 7 identified isoforms . It functions primarily as an integral membrane protein that regulates neurotransmitter release in the central nervous system .

HRH3 plays critical roles in multiple physiological processes, including:

• Modulation of chemical synaptic transmission

• Regulation of histamine levels and neurotransmitter release

• Influence on cognitive processes, sleep-wake cycles, and appetite control

• Increase of voltage-dependent calcium current in smooth muscles

• Innervation of blood vessels and the heart in the cardiovascular system

These diverse functions make HRH3 an important target for both basic research and potential therapeutic development.

What are the primary applications for HRH3 antibodies in research?

HRH3 antibodies serve multiple experimental purposes across different methodologies:

• Western Blot (WB): The most common application, used for protein detection and quantification in cell and tissue lysates

• ELISA: For quantitative measurement of HRH3 in solution

• Immunohistochemistry (IHC): To visualize HRH3 distribution in tissue sections

• Flow Cytometry (FC): For analysis of HRH3 expression at the cellular level, typically using dilutions in the 1:50-1:200 range

• Immunoprecipitation (IP): For isolation of HRH3 protein complexes

Each application requires specific optimization protocols to ensure reliable and reproducible results. Researchers should determine optimal dilutions experimentally for their particular biological system and antibody preparation .

How are HRH3 recombinant monoclonal antibodies produced?

The production of HRH3 recombinant monoclonal antibodies employs sophisticated biotechnology methods:

• Initial immunization typically uses synthesized peptides derived from human HRH3 protein sequences

• Antibody-encoding DNA sequences are cloned from immunoreactive rabbits

• These genes are inserted into plasmid vectors for recombinant expression

• Host cells are transfected with these vectors to enable antibody production

• Following expression, the antibodies undergo purification through affinity chromatography, typically using Protein A or G columns

• Quality control testing confirms reactivity with human HRH3 protein using applications such as ELISA and flow cytometry

This recombinant approach offers significant advantages over traditional hybridoma-based methods, including improved batch-to-batch consistency and reduced dependence on animal immunization procedures .

What factors should be considered when validating HRH3 antibody specificity?

Proper validation is critical for ensuring experimental reliability. For HRH3 antibodies, comprehensive validation should include:

• Positive control testing using:

  • HEK293 cells transfected with human HRH3

  • Tissues known to express HRH3 (brain regions, particularly cerebral cortex)

• Negative control analysis:

  • HEK293 cells transfected with irrelevant proteins

  • Primary antibody omission

  • Isotype-matched control antibodies

• Reactivity confirmation:

  • Testing across multiple species if working with human/mouse/rat reactive antibodies

  • Verification with synthetic peptide competition assays

• Application-specific validation:

  • For flow cytometry: proper compensation and fluorescence-minus-one controls

  • For Western blot: confirmation of expected molecular weight (~48.7 kDa)

Thorough validation minimizes the risk of experimental artifacts and ensures scientific reproducibility.

What are the optimal conditions for using HRH3 antibodies in Western blot applications?

For optimal Western blot results with HRH3 antibodies, researchers should consider:

• Sample preparation:

  • Proper lysis buffers containing detergents appropriate for membrane proteins

  • Fresh protease inhibitors to prevent degradation

  • Careful temperature control during preparation

• Electrophoresis parameters:

  • 10-12% SDS-PAGE gels typically provide good resolution for the ~48.7 kDa HRH3 protein

  • Include positive controls (e.g., HRH3-transfected cell lysates)

• Transfer conditions:

  • PVDF membranes often work better than nitrocellulose for membrane proteins

  • Transfer at lower voltage for longer time to ensure complete transfer

• Antibody incubation:

  • Proper blocking (typically 5% BSA or non-fat milk)

  • Optimized antibody dilution (starting recommendations vary by product)

  • Extended incubation times at 4°C can improve signal quality

• Special considerations:

  • HRH3 can form dimers or undergo post-translational modifications, potentially resulting in multiple bands

  • Membrane proteins may require specialized handling to prevent aggregation

How can researchers optimize HRH3 detection in flow cytometry experiments?

Flow cytometry with HRH3 antibodies requires careful preparation and execution:

• Sample preparation:

  • Gentle cell dissociation methods to preserve membrane integrity

  • Fixation protocols that maintain antigen structure (typically 2-4% paraformaldehyde)

  • Appropriate permeabilization for intracellular domains (if applicable)

• Antibody selection and application:

  • Direct-conjugated antibodies (e.g., Alexa Fluor® 647 or APC/Cy7) for multicolor panels

  • Dilution optimization (recommended starting range: 1:50-1:200)

  • Adequate incubation time (typically 30-60 minutes on ice)

• Technical optimization:

  • Proper control samples (unstained, isotype, FMO)

  • Protection from light for fluorophore-conjugated antibodies

  • Avoidance of temperature fluctuations that could lead to degradation or decoupling of tandem dyes

• Analysis considerations:

  • Use of appropriate gating strategies based on control samples

  • Accounting for potential autofluorescence from certain cell types

  • Proper compensation when using multiple fluorophores

Researchers should consult with technical support if experiencing difficulties with specific antibody preparations .

How can HRH3 antibodies be used to study receptor localization in neuronal tissues?

HRH3 antibodies enable detailed investigation of receptor distribution in neural circuits:

• Immunohistochemical approaches:

  • Fixed tissue sections from brain regions of interest

  • Optimization of antigen retrieval methods for better epitope access

  • Co-staining with neuronal and glial markers to determine cell-type specific expression

• Subcellular localization studies:

  • Co-localization with synaptic markers to assess pre- versus post-synaptic distribution

  • Electron microscopy for ultrastructural localization

  • Super-resolution microscopy techniques for precise spatial mapping

• Functional correlation:

  • Combining immunostaining with electrophysiological recordings

  • Analysis of receptor distribution in relation to neurotransmitter systems

  • Examination of changes in receptor localization under different physiological states

These approaches provide valuable insights into how HRH3 functions within specific neural circuits and its potential role in neurological and psychiatric disorders.

What considerations are important when studying HRH3 in disease models?

Investigating HRH3 in pathological conditions requires special methodological attention:

• Model selection and validation:

  • Appropriate animal models relevant to HRH3-associated pathologies (neurological, cardiovascular)

  • Verification of HRH3 expression and conservation of relevant signaling pathways

  • Control for potential species differences in receptor pharmacology and distribution

• Experimental design:

  • Temporal analysis to track receptor changes throughout disease progression

  • Inclusion of age-matched and treatment controls

  • Correlation of HRH3 changes with functional/behavioral outcomes

• Technical approaches:

  • Quantitative analysis methods (e.g., Western blot densitometry, quantitative immunohistochemistry)

  • Multiplexed analysis to examine HRH3 in relation to other disease markers

  • Functional assays to assess receptor activity, not just expression levels

• Translational considerations:

  • Validation of findings across multiple model systems

  • Comparison with human tissue samples when available

  • Correlation of antibody-based findings with genetic and pharmacological studies

These methodological considerations help ensure that HRH3-focused research yields results with potential clinical relevance.

What are common challenges when working with HRH3 antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when using HRH3 antibodies:

• Low signal intensity:

  • Increase antibody concentration (within manufacturer guidelines)

  • Extend incubation time (e.g., overnight at 4°C)

  • Use signal amplification systems (e.g., biotin-streptavidin)

  • Optimize antigen retrieval methods for tissue sections

• High background:

  • Increase washing steps (frequency and duration)

  • Optimize blocking conditions (try different blocking agents)

  • Reduce antibody concentration

  • Pre-absorb antibody with non-specific proteins

• Non-specific binding:

  • Validate antibody specificity with appropriate controls

  • Use more stringent washing conditions

  • Consider different fixation methods that better preserve epitope structure

  • Test alternative antibody clones targeting different epitopes

• Batch-to-batch variation:

  • Standardize protocols with detailed documentation

  • Consider recombinant monoclonal antibodies for better consistency

  • Validate each new lot against previous successful experiments

Careful optimization and systematic troubleshooting are essential for obtaining reliable results with HRH3 antibodies.

How can storage and handling practices affect HRH3 antibody performance?

Proper storage and handling are critical for maintaining antibody functionality:

• Storage recommendations:

  • Store lyophilized antibodies at -20°C for long-term stability

  • After reconstitution, store at 4°C for short-term use (typically one month)

  • For conjugated antibodies, protect from light and never freeze

  • Avoid repeated freeze-thaw cycles

• Handling best practices:

  • Maintain cold chain during experimental procedures

  • Prepare small working aliquots to minimize freeze-thaw cycles

  • Use sterile technique when handling antibody solutions

  • Follow manufacturer-specific recommendations for each preparation

• Special considerations for conjugated antibodies:

  • Fluorophore-conjugated antibodies require protection from light

  • Tandem dyes are particularly sensitive to temperature fluctuations and light exposure

  • Some conjugates have specific storage requirements that differ from unconjugated antibodies

• Quality control:

  • Document lot numbers and performance

  • Test new antibody preparations against established positive controls

  • Consider including stability controls in long-term studies

Proper storage and handling significantly extend antibody shelf-life and ensure consistent experimental results.

How might advanced imaging techniques enhance HRH3 antibody applications?

Emerging imaging technologies offer new possibilities for HRH3 research:

• Super-resolution microscopy:

  • Single-molecule localization methods (STORM, PALM) for nanoscale receptor distribution

  • Structured illumination microscopy (SIM) for improved resolution without specialized probes

  • Expansion microscopy techniques to physically enlarge specimens for better visualization

• Multiplexed imaging approaches:

  • Cyclic immunofluorescence for simultaneous detection of dozens of markers

  • Mass cytometry imaging for highly multiplexed tissue analysis

  • Spatial transcriptomics coupled with protein detection

• Live-cell imaging technologies:

  • Development of non-disruptive labeling strategies

  • CRISPR-based tagging systems for endogenous HRH3 visualization

  • Single-particle tracking to monitor receptor dynamics

• Computational analysis methods:

  • Machine learning algorithms for automated detection and quantification

  • 3D reconstruction and modeling of receptor distribution

  • Systems biology approaches integrating spatial and functional data

These advanced technologies promise to reveal HRH3 distribution and dynamics with unprecedented detail and functional context.

What emerging therapeutic applications might benefit from HRH3 antibody research?

HRH3 antibody research supports several promising therapeutic directions:

• Neurological applications:

  • Cognitive disorders and dementia, leveraging HRH3's role in neurotransmitter regulation

  • Sleep disorders, based on HRH3's involvement in sleep-wake cycles

  • Neuroinflammatory conditions where histamine signaling plays a modulatory role

• Metabolic disorders:

  • Obesity research, given HRH3's influence on appetite regulation

  • Metabolic syndrome and related conditions

• Cardiovascular indications:

  • Targeting HRH3's effects on blood vessels and cardiac function

  • Regulation of calcium currents in vascular smooth muscle

• Precision medicine approaches:

  • Development of companion diagnostics for HRH3-targeting therapeutics

  • Patient stratification based on receptor expression profiles

  • Monitoring therapeutic response through receptor occupancy studies

Antibody-based research provides critical insights that can guide drug development and patient selection for clinical trials targeting the HRH3 receptor system.