HRH2 Antibody

What is the HRH2 receptor and what are its key structural characteristics?

The Histamine H2 Receptor (HRH2) is a G protein-coupled receptor (GPCR) involved in chemical synaptic transmission and immune response pathways. The human version has a canonical amino acid length of 359 residues and a protein mass of 40.1 kilodaltons, although two isoforms have been identified. HRH2 is localized in the cell membrane and is notably expressed in the duodenum and small intestine .

For researchers performing transcript analysis, it's important to note that HRH2 has two transcript variants:

• Variant 1 (ENST00000377291): A 2561 base pair transcript with 2 retained introns and 3 exons, coding for a 397 amino acid protein

• Variant 2 (ENST00000231683): A lower abundance, 1080 base pair intronless transcript coding for a 359 amino acid protein (considered the canonical sequence)

What applications are HRH2 antibodies commonly used for?

HRH2 antibodies are used in multiple applications to detect and measure the Histamine H2 Receptor in biological samples:

Western Blot is the most widely used application, frequently complemented by ELISA for quantification purposes .

How should I select the appropriate HRH2 antibody for my specific research question?

Selection of an appropriate HRH2 antibody depends on several critical factors:

• Target region specificity: Different antibodies target different regions of HRH2:

  • N-terminal region antibodies

  • C-terminal region antibodies

  • Extracellular domain antibodies

  • Second extracellular loop antibodies (e.g., amino acids 161-173)

  • Internal region antibodies

• Species reactivity: Confirm cross-reactivity with your experimental species. Available antibodies show reactivity with:

  • Human only

  • Human and mouse

  • Human, mouse, and rat

  • Multiple species including dog and horse

• Application compatibility: Not all antibodies work equally well across applications. Review validation data for your specific application (WB, IF, IHC, etc.) .

• Host species: Consider the host species (typically rabbit or goat) in relation to your experimental design, especially if performing multi-labeling experiments .

• Clonality: Polyclonal antibodies generally provide higher sensitivity but may have more batch-to-batch variation compared to monoclonal antibodies .

What are the optimal conditions for Western blot detection of HRH2?

For successful Western blot detection of HRH2:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors to prevent protein degradation.

• Expected molecular weight: While the calculated molecular weight of HRH2 is approximately 40-45 kDa, the observed molecular weight is typically higher:

  • 59 kDa and 69 kDa bands are commonly observed

  • In rat basophilic leukemia cells and rat brain lysates, distinct bands can be identified

• Dilution ranges: Most antibodies work optimally at dilutions between 1:500-1:1000 for Western blot .

• Blocking conditions: 5% non-fat milk or BSA in TBST is typically effective.

• Validation control: Use positive control samples such as:

  • SH-SY5Y cells

  • Human skeletal muscle tissue

  • Mouse stomach tissue

• Preincubation control: For specificity verification, perform parallel experiments with the antibody preincubated with the immunizing peptide .

How can HRH2 antibodies be employed to study receptor localization in tissues?

For effective receptor localization studies:

• Immunohistochemistry approach: Use paraffin-embedded sections with appropriate antigen retrieval methods. For example, in rat stomach sections, HRH2 antibodies have successfully demonstrated expression in parietal cells of gastric glands using 1:100 dilution .

• Cell surface detection: For live intact cell studies:

  • Use antibodies targeting extracellular epitopes

  • Implement non-permeabilizing conditions

  • Apply secondary antibodies conjugated with fluorophores such as FITC

• Subcellular localization:

  • Use confocal microscopy with markers for cellular compartments

  • Compare membrane versus cytoplasmic distribution

  • Consider co-localization with signaling partners

• Validation controls:

  • Include peptide blocking controls

  • Use tissues known to express high levels of HRH2 (duodenum, small intestine)

  • Include negative control tissues with low expression

What approaches can be used to study HRH2 polymorphisms and their association with disease?

Based on research studying HRH2 polymorphisms and heart failure:

• SNP identification strategy:

  • Target the 28 kilobase region of HRH2 located on chromosome 5q35.2

  • Consider both coding and non-coding regions including introns

  • Use platforms such as Illumina GoldenGate Assay for genotyping

• Statistical analysis approaches:

  • Apply Cox proportional hazards regression for association with disease incidence

  • Adjust for appropriate demographic and clinical covariates

  • Consider ethnicity-specific analyses (as demonstrated in the Multi-Ethnic Study of Atherosclerosis)

• Functional validation:

  • Analyze mRNA expression using RNA-Seq to identify transcript variants

  • Quantify using Fragments per kilobase per transcript per million mapped reads (FPKM)

  • Compare variant expression between phenotypic groups

• Key findings from existing research:

  • The rs2241562 minor allele was associated with increased heart failure incidence in Chinese participants (adjusted hazard ratio 3.7, 95% CI 1.0 to 13.4)

  • Super-responders to beta-blockade had higher baseline myocardial HRH2 transcript levels compared to non-responders

What are common challenges in HRH2 antibody experiments and how can they be addressed?

• Multiple band detection:

  • Expected observation: Multiple bands may represent different isoforms or post-translational modifications

  • Solution: Use specific positive controls and blocking peptides to confirm specificity

  • Verification: Compare observed bands (59 kDa, 69 kDa) with literature reports

• Tissue-specific expression variations:

  • Challenge: HRH2 expression varies significantly between tissues

  • Approach: Adjust antibody concentration based on expected expression level

  • Controls: Include tissues with known high expression (stomach, duodenum)

• Species cross-reactivity:

  • Issue: Some antibodies show limited cross-species reactivity

  • Solution: Choose antibodies with validated reactivity for your species

  • Example: For rat studies, confirm antibody reactivity percentage (e.g., Rat: 85%)

• Fixation sensitivity:

  • Problem: Some epitopes may be sensitive to certain fixatives

  • Optimization: Test multiple fixation protocols if initial results are suboptimal

  • Alternative: Consider live cell labeling for surface epitopes

How can I validate the specificity of an HRH2 antibody?

For rigorous antibody validation:

• Peptide competition assay:

  • Preincubate antibody with immunizing peptide

  • Compare signal between blocked and unblocked antibody

  • Loss of signal indicates specificity

• Positive control samples:

  • Use tissues/cells with known HRH2 expression:

    • Rat basophilic leukemia cells

    • SH-SY5Y cells

    • Stomach tissue

    • Duodenum tissue

• Multiple application validation:

  • Test antibody across different applications (WB, IHC, IF)

  • Consistent results across applications increase confidence

  • Compare with published literature results

• Molecular weight verification:

  • Compare observed bands with expected molecular weights

  • Account for post-translational modifications

  • Verify with different antibodies targeting distinct epitopes

What is the role of HRH2 in cardiac function and how can antibodies help investigate this?

Research has indicated HRH2's involvement in cardiac function:

• Experimental evidence:

  • H2 receptor activation contributes to heart failure in preclinical models

  • H2 receptor antagonists are associated with decreased heart failure incidence

• Transcript analysis approach:

  • Collect myocardial biopsies (e.g., right ventricular distal septum)

  • Extract RNA and perform RNA-Seq

  • Quantify HRH2 transcript variants (Variant 1 and Variant 2)

  • Compare expression between phenotypic groups

• Key findings in dilated cardiomyopathy:

  • Super-responders to beta-blockade showed higher baseline levels of myocardial HRH2 transcripts compared to non-responders

  • Variant 2: 5.5 ±1.1 FPKM in super-responders vs. 3.2 ±0.8 FPKM in non-responders (p=0.002)

  • Total HRH2 (Variant 1+2): 32.1 ±7.4 FPKM in super-responders vs. 23.3 ±4.2 FPKM in non-responders (p=0.04)

• Antibody applications:

  • Immunohistochemistry to localize receptor in cardiac tissue

  • Western blotting to quantify protein expression changes

  • Immunoprecipitation to identify interaction partners

How can immunohistochemical techniques be optimized for HRH2 detection in tissue sections?

For optimal immunohistochemical detection:

• Tissue preparation:

  • Fixation: 10% neutral buffered formalin or 4% paraformaldehyde

  • Embedding: Paraffin embedding preserves tissue architecture

  • Sectioning: 4-5 μm sections typically provide optimal results

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Enzymatic retrieval with proteases for certain epitopes

  • Optimization based on specific antibody recommendations

• Protocol optimization:

  • Antibody dilution: Start with 1:100 as demonstrated in rat stomach sections

  • Incubation time: Typically overnight at 4°C or 1-2 hours at room temperature

  • Detection system: HRP/DAB or fluorescent secondary antibodies

  • Counterstain: Hematoxylin provides good nuclear contrast

• Expression patterns:

  • In gastric tissue: HRH2 is expressed in parietal cells of gastric glands

  • Cellular pattern: Predominantly membrane localization with some cytoplasmic staining

  • Use appropriate positive controls based on known expression patterns

What emerging techniques might enhance HRH2 receptor research?

Several emerging approaches could advance HRH2 research:

• Single-cell analysis techniques:

  • Single-cell RNA-seq to characterize cell-specific expression patterns

  • Mass cytometry (CyTOF) for high-dimensional protein analysis

  • Spatial transcriptomics to map receptor expression in tissue context

• CRISPR/Cas9 applications:

  • Generate knockout models to study receptor function

  • Create epitope-tagged endogenous receptors for improved detection

  • Introduce specific polymorphisms to study functional consequences

• Advanced imaging approaches:

  • Super-resolution microscopy for nanoscale receptor organization

  • Intravital imaging to study receptor dynamics in vivo

  • Correlative light and electron microscopy for ultrastructural localization

• Therapeutic targeting strategies:

  • Development of monoclonal antibodies for receptor modulation

  • Investigation of novel H2 receptor antagonists with improved specificity

  • Exploration of potential biased signaling modulators

By combining these advanced methodological approaches with existing antibody-based techniques, researchers can gain deeper insights into HRH2 biology and its role in health and disease.