HTR3E Antibody,HRP conjugated

What is HTR3E and what biological role does it play in human physiology?

HTR3E is a subunit E of the type 3 receptor for 5-hydroxytryptamine (serotonin), a biogenic hormone that functions as a neurotransmitter, hormone, and mitogen. It belongs to the ligand-gated ion channel receptor superfamily and causes fast, depolarizing responses in neurons after activation. HTR3E is part of a gene cluster on chromosome 3 that includes subunits C and D, with alternative splice variants identified though not fully characterized . The HTR3E subunit combines with other 5-HT3 subunits, particularly 5-HT3A, to form functional heteromeric receptors that contribute to serotonergic signaling in various tissues, especially within the gastrointestinal system .

What is the molecular basis for HTR3E antibody specificity and what epitopes are typically targeted?

HTR3E antibodies are typically generated against specific regions of the protein, with commercial antibodies often targeting the central/middle region (amino acids 133-162) or other defined sequences (AA 126-154, AA 26-248, or AA 360-409) . Specificity is achieved through careful immunogen design, commonly using KLH-conjugated synthetic peptides from human 5HT3E sequences. Validation typically involves detection of correctly sized bands in Western blots from transfected cells expressing the target protein (~55 kDa for HTR3E) and demonstration of signal abolishment through peptide competition assays .

What are the recommended protocols for using HTR3E antibody, HRP conjugated in ELISA experiments?

For ELISA applications using HTR3E antibody, HRP conjugated, the following protocol guidelines are recommended:

• Coat plates with capture antigen (recombinant HTR3E or tissue lysate) at 1-10 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C

• Block with 3-5% BSA or non-fat milk in PBST for 1-2 hours at room temperature

• Incubate with HTR3E antibody, HRP conjugated at 1:100-1:1000 dilution in blocking buffer for 1-2 hours at room temperature

• Wash 4-5 times with PBST

• Develop with TMB substrate and stop with H₂SO₄

• Read absorbance at 450nm

Optimization should include antibody titration experiments to determine the optimal signal-to-noise ratio for your specific sample type .

How should Western blotting protocols be adapted when using the HRP-conjugated form of HTR3E antibody?

When using HTR3E antibody, HRP conjugated for Western blotting:

• Prepare protein samples with reducing buffer and heat at 95°C for 5 minutes

• Separate proteins by SDS-PAGE (10-12% gel recommended)

• Transfer to PVDF or nitrocellulose membrane

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with HTR3E antibody, HRP conjugated at 1:500-1:1000 dilution in blocking buffer overnight at 4°C

• Wash 3-4 times with TBST, 5-10 minutes each

• Develop directly with enhanced chemiluminescence substrate

Note that unlike non-conjugated antibodies, no secondary antibody incubation is required. Expected molecular weight for HTR3E is approximately 55 kDa. Researchers may need to optimize exposure times to achieve ideal signal intensity without background .

What sample preparation methods maximize detection sensitivity for HTR3E in complex tissue samples?

For optimal detection of HTR3E in tissue samples:

• Fresh tissue samples should be rapidly fixed in 4% paraformaldehyde or flash-frozen

• For protein extraction, use RIPA buffer supplemented with protease inhibitors

• Homogenize tissues thoroughly using mechanical disruption

• Clarify lysates by centrifugation at 14,000g for 15 minutes at 4°C

• Determine protein concentration using BCA or Bradford assay

• Load 20-50 μg of total protein per lane for Western blotting

• For immunohistochemistry, consider antigen retrieval with TE buffer (pH 9.0) as this has been shown to enhance detection of serotonin receptor family members

HTR3E is particularly enriched in gastrointestinal tissues, especially in myenteric neurons, which should be considered when selecting sample sources .

How can researchers differentiate between HTR3E and other 5-HT3 receptor subunits in experimental systems?

Differentiating between 5-HT3 receptor subunits requires careful experimental design:

• Select antibodies with confirmed specificity for HTR3E with minimal cross-reactivity to other subunits

• Use multiple antibodies targeting different epitopes of HTR3E to confirm findings

• Include appropriate positive controls (transfected cells expressing only HTR3E) and negative controls

• Consider co-immunoprecipitation experiments to assess subunit interactions

• Complement protein detection with mRNA analysis using subunit-specific primers

• For functional studies, use pharmacological agents with differential selectivity for receptor subtypes

Research has demonstrated that 5-HT3C, 5-HT3D, and 5-HT3E subunits are coexpressed with 5-HT3A in cell bodies of myenteric neurons, requiring careful experimental design to distinguish individual subunit contributions .

What experimental approaches can validate the specificity of HTR3E antibody in different applications?

Rigorous validation of HTR3E antibody specificity should include:

• Western blot analysis showing bands of expected molecular weight (~55 kDa)

• Peptide competition assays demonstrating signal abolishment when antibody is pre-incubated with immunogenic peptide

• Testing in cell lines with confirmed HTR3E expression (e.g., HEK293 or HT-29 cells transfected with HTR3E)

• Knockdown experiments using siRNA targeting HTR3E to confirm signal reduction

• Parallel testing with multiple antibodies targeting different epitopes

• Cross-validation with mRNA expression data

Previous research has validated antibody specificity through immunoprecipitation experiments using lysates of HEK293 cells expressing HTR3E subunits, with immunoreactive bands at the expected size of ~55 kDa .

How should researchers interpret differential expression patterns of HTR3E in disease models versus healthy tissues?

When analyzing HTR3E expression differences between disease and healthy states:

• Quantify expression using multiple methodologies (qPCR, Western blot, immunohistochemistry)

• Normalize expression data to appropriate housekeeping genes/proteins

• Consider post-translational modifications that may affect antibody recognition

• Evaluate expression in context of other 5-HT3 receptor subunits

• Correlate findings with functional outcomes in disease models

• Examine potential regulatory mechanisms (e.g., miRNA regulation)

Research has shown increased HTR3E expression in colonic mucosal tissue of patients with diarrhea-predominant irritable bowel syndrome (IBS-D) compared to controls, associated with decreased miR-510 expression. Additionally, HTR3E expression was significantly higher in patients with the GA genotype (rs56109847) compared to those with the GG genotype .

What are the common technical challenges when using HTR3E antibody, HRP conjugated, and how can they be addressed?

Common challenges and solutions include:

Optimization should be performed systematically, changing one variable at a time .

How can researchers quantitatively analyze HTR3E expression data across different experimental conditions?

For quantitative analysis of HTR3E expression:

• For Western blots:

  • Use image analysis software (ImageJ, Image Lab)

  • Normalize band intensity to loading controls (β-actin, GAPDH)

  • Include standard curves with known quantities when possible

  • Analyze 3-5 independent biological replicates

• For ELISA:

  • Generate standard curves using recombinant HTR3E protein

  • Ensure measurements fall within linear range of detection

  • Run samples in technical triplicates

  • Apply appropriate statistical tests (t-test, ANOVA)

• For RT-PCR:

  • Use validated reference genes for normalization

  • Apply 2^(-ΔΔCt) method for relative quantification

  • Validate primers for specificity and efficiency

Include appropriate statistical analysis and report both mean values and measures of variance .

What considerations are important when investigating HTR3E in the context of genetic variations like rs56109847?

When studying genetic variations affecting HTR3E:

• Design genotyping methods that reliably distinguish variants:

  • PCR followed by restriction enzyme digestion (e.g., Hpy188III for rs56109847)

  • Confirm genotypes through nucleotide sequencing

  • Use appropriate controls for each genotype

• For functional analysis of variants:

  • Construct expression vectors containing variant sequences

  • Use dual-luciferase reporter assays to quantify effects

  • Transfect into relevant cell lines (HEK293, HT-29)

  • Include appropriate controls (empty vectors, irrelevant miRNAs)

• Correlate genotype with phenotype:

  • Consider population stratification

  • Adjust for covariates in statistical analysis

  • Validate findings in independent cohorts

Research has demonstrated that the rs56109847 variant disrupts the binding site of miR-510 and significantly upregulates luciferase expression in HEK293 and HT-29 cells, potentially explaining its association with IBS-D .

How can HTR3E antibodies be effectively employed in multiplexed immunoassays with other serotonin receptor antibodies?

For multiplexed detection of serotonin receptor subunits:

• Select antibodies raised in different host species to enable simultaneous detection

• Alternatively, use directly conjugated antibodies with different fluorophores or enzymes

• Validate absence of cross-reactivity between antibodies

• Optimize signal-to-noise ratio for each antibody individually before multiplexing

• Consider spectral overlap when selecting fluorophores

• Include appropriate single-stained controls for compensation in flow cytometry

• For immunohistochemistry, use sequential staining protocols with thorough washing between antibodies

This approach is particularly valuable for studying coexpression patterns of HTR3A, HTR3C, HTR3D, and HTR3E in tissues like the human colon, where these subunits have been shown to colocalize in myenteric neurons .

What experimental approaches can elucidate the functional significance of HTR3E in serotonergic signaling pathways?

To investigate HTR3E functional significance:

• Design gene silencing experiments:

  • Use siRNA or CRISPR-Cas9 to knock down HTR3E

  • Validate knockdown efficiency at protein and mRNA levels

  • Assess functional consequences on calcium flux, membrane potential, or cell signaling

• Develop pharmacological approaches:

  • Use subtype-selective agonists/antagonists

  • Perform dose-response studies

  • Measure functional outputs (electrophysiology, calcium imaging)

• Analyze protein-protein interactions:

  • Co-immunoprecipitation of HTR3E with other subunits

  • Proximity ligation assays to detect in situ interactions

  • FRET/BRET analysis for real-time interaction studies

• Investigate trafficking and localization:

  • Use confocal microscopy with subcellular markers

  • Perform surface biotinylation assays

  • Analyze receptor internalization dynamics

These approaches can help determine how HTR3E contributes to receptor function, particularly in heteromeric complexes with HTR3A .

How can researchers design experiments to investigate the therapeutic potential of targeting HTR3E in gastrointestinal disorders?

For investigating HTR3E as a therapeutic target:

• Design preclinical studies:

  • Select appropriate disease models (IBS-D animal models, organoids)

  • Evaluate HTR3E expression in diseased vs. normal tissues

  • Assess correlation between HTR3E expression/function and disease phenotypes

• Develop targeting strategies:

  • Screen for HTR3E-selective compounds

  • Design antibodies for receptor modulation rather than detection

  • Explore gene therapy approaches to modulate expression

• Establish functional readouts:

  • Measure gastrointestinal motility (transit assays)

  • Evaluate secretory functions (Ussing chamber)

  • Assess pain sensitivity in animal models

  • Quantify inflammatory markers

• Investigate genetic associations:

  • Expand on known associations like rs56109847

  • Perform genotype-phenotype correlations

  • Design personalized therapeutic approaches based on genetic profiles