HTR3E Antibody，Biotin conjugated

What is HTR3E and what are its functional properties in biological systems?

HTR3E encodes subunit E of the type 3 receptor for 5-hydroxytryptamine (serotonin), belonging to the ligand-gated ion channel receptor superfamily. This protein functions as part of a serotonin-activated cation-selective channel complex, which when activated causes fast, depolarizing responses in neurons. HTR3E is primarily expressed in adult colon and intestine tissues, and forms part of a gene cluster with subunits C and D on chromosome 3. The protein has a calculated molecular weight of approximately 51.4 kDa and functions at the postsynaptic cell membrane as a multi-pass membrane protein. Notably, HTR3E is presumably retained within the endoplasmic reticulum unless complexed with HTR3A, indicating its dependency on partner proteins for proper localization and function .

Research has shown that HTR3E along with HTR3C and HTR3D cannot form functional 5-HT3 receptors independently but require co-expression with the HTR3A subunit to create functional heteromeric receptors with distinctive pharmacological properties. These combinations create receptor subtypes that differ in their maximal responses to serotonin, potentially explaining the heterogeneity of 5-HT3 receptor responses observed in various tissues .

What is the significance of biotin conjugation in HTR3E antibodies?

Biotin conjugation provides significant advantages for research applications through the exceptionally strong non-covalent interaction between biotin and streptavidin proteins. For HTR3E antibodies, this conjugation enables:

• Enhanced sensitivity through signal amplification, as multiple streptavidin molecules can bind to each biotin-conjugated antibody

• Compatibility with diverse detection systems using streptavidin conjugated to various reporter molecules (fluorophores, enzymes)

• Increased flexibility in experimental design, particularly for multi-step staining protocols

The conjugation process typically involves NHS-Biotin reacting with primary amines on the antibody structure. A standard protocol uses a 5:1 molar ratio of biotinylating reagent to antibody, with the reaction occurring under constant stirring at room temperature for approximately 30 minutes. Purification steps using protein A affinity chromatography remove unreacted biotin and ensure antibody activity is preserved .

This modification is particularly valuable for applications requiring high sensitivity or when working with samples containing low levels of HTR3E expression, as the biotin-streptavidin system provides significant signal enhancement compared to direct detection methods.

What applications are most suitable for biotin-conjugated HTR3E antibodies?

Based on vendor specifications and research literature, biotin-conjugated HTR3E antibodies are optimized for several specific applications:

While non-conjugated HTR3E antibodies are frequently used in Western blotting applications, biotin-conjugated versions are particularly advantageous for ELISA applications as noted in multiple product specifications . Research demonstrates that biotin-conjugated antibodies against 5-HT3 receptor subunits have been successfully employed in immunofluorescence studies examining serotonin receptor distribution in the human colon, where they allowed for detailed localization of receptor subunits in myenteric neurons .

How should researchers optimize sample preparation for HTR3E antibody detection?

Optimal sample preparation is crucial for successful detection of HTR3E proteins. Based on published methodologies, researchers should consider:

For cellular samples:

• Fixation: Use 4% paraformaldehyde for 10-15 minutes at room temperature for cultured cells, maintaining membrane integrity while preserving epitope accessibility.

• Permeabilization: Apply 0.1% Triton X-100 for 5-10 minutes to allow antibody access to intracellular epitopes, particularly important as HTR3E may be retained in the endoplasmic reticulum before complex formation.

• Blocking: Implement a thorough blocking step (3-5% BSA or 10% normal serum) for at least 30 minutes to reduce non-specific binding.

For tissue sections:

• Tissue processing: For colon or intestinal tissues (where HTR3E is natively expressed), optimal fixation and sectioning at 5-10μm thickness ensures antibody penetration.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for formalin-fixed tissues to unmask antigens potentially obscured during fixation.

• Endogenous biotin blocking: Critical for intestinal tissues which contain high levels of endogenous biotin; use avidin/biotin blocking kits prior to antibody application to prevent false positive results .

For protein extracts:

• Extraction buffers: Use buffers containing mild detergents (0.5-1% NP-40 or Triton X-100) with protease inhibitors to preserve protein integrity.

• Protein quantification: Standardize protein loading across samples for consistent comparisons of HTR3E expression levels.

What validation strategies confirm HTR3E antibody specificity?

Rigorous validation is essential for ensuring reliable results. Based on published approaches, researchers should implement multiple validation strategies:

• Recombinant protein expression systems:

  • Transfect cells (HEK293, COS7) with expression constructs for HTR3E

  • Compare antibody reactivity between transfected and non-transfected cells

  • Detect bands of expected molecular weight (~51kDa) in Western blot analysis

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide (100μg)

  • Verify elimination of specific staining in immunofluorescence experiments

  • Confirm abolishment of Western blot signals at the expected molecular weight

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of HTR3E

  • Compare staining patterns for consistency

  • Employ both monoclonal and polyclonal antibodies when available

• RNA-protein correlation:

  • Perform RT-PCR on microdissected tissue samples to detect transcripts

  • Correlate mRNA expression with protein detection patterns

In published studies, researchers confirmed antibody specificity by demonstrating that "specific signal was abolished after preadsorption with 100 μg of the immunogen" and preincubation with peptide immunogen "abolished the staining in immunofluorescence experiments," providing conclusive evidence of specificity .

What detection systems maximize sensitivity with biotin-conjugated HTR3E antibodies?

Several detection systems can be employed to maximize sensitivity when using biotin-conjugated HTR3E antibodies:

• Streptavidin-conjugated reporter systems:

  • Streptavidin-HRP: Optimal for ELISA and chromogenic IHC with 3,3'-diaminobenzidine (DAB)

  • Streptavidin-fluorophores: Available with various fluorescent dyes (Alexa Fluor 488, Cy3, Cy5) for multicolor imaging

  • Streptavidin-gold: For electron microscopy applications to define subcellular localization

• Signal amplification methods:

  • Tyramide Signal Amplification (TSA): Can increase sensitivity 10-100 fold over conventional methods

  • ABC (Avidin-Biotin Complex): Utilizes multiple biotin binding sites on avidin molecules

  • Streptavidin-poly-HRP: Provides multiple HRP molecules per binding event for enhanced signal

• Detection optimization parameters:

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

  • Washing steps: Use PBS-T (0.05-0.1% Tween-20) with sufficient volume and duration

  • Signal development: Optimize timing for chromogenic substrates to maximize signal-to-noise ratio

Research protocols have demonstrated successful use of fluorophore-labeled detection systems including "N-hydroxysuccinimide-activated fluorescein for 5-HT3C, 5-HT3D, and 5-HT3E, 5(6)-carboxytetramethylrhodamine for 5-HT3A, and Cy3B for 5-HT3C, 5-HT3D, and 5-HT3E" in immunofluorescence studies, enabling detailed visualization of receptor subunit localization .

How can HTR3E antibodies be employed in multiplex detection systems?

Multiplex detection enables simultaneous visualization of multiple targets, providing valuable contextual information about HTR3E expression and colocalization with other proteins:

• Multicolor immunofluorescence strategies:

  • Use biotin-conjugated HTR3E antibody with streptavidin-fluorophore (e.g., Cy3)

  • Combine with directly labeled antibodies against other targets using spectrally distinct fluorophores

  • Apply sequential staining if antibodies are raised in the same species, using blocking steps between applications

• Implementation example from literature:
  Research has successfully demonstrated multiplex detection of different 5-HT3 receptor subunits using:

  • Fluorescein-labeled antibodies against 5-HT3C, 5-HT3D, and 5-HT3E

  • Carboxytetramethylrhodamine-labeled antibodies against 5-HT3A

  • This approach enabled colocalization studies showing that "5-HT3C, 5-HT3D, and 5-HT3E subunits are coexpressed with 5-HT3A in cell bodies of myenteric neurons"

• Sequential multiplex protocol:

  • First round: Apply biotin-conjugated HTR3E antibody, detect with streptavidin-fluorophore

  • Document images with precise coordinates

  • Elution step: Use glycine buffer (pH 2.0) or commercial antibody stripping solutions

  • Second round: Apply antibodies against additional targets, detect with appropriate systems

  • Image registration: Align images from multiple rounds to create composite visualization

• Controls for multiplex experiments:

  • Single-staining controls to assess spectral overlap

  • Absorption controls using immunizing peptides for each target

  • Secondary-only controls to evaluate non-specific binding

What experimental approaches can determine the subcellular localization of HTR3E?

Determining subcellular localization provides insights into receptor trafficking, processing, and functional state:

• High-resolution imaging techniques:

  • Confocal microscopy: For optical sectioning and 3D reconstruction

  • Super-resolution microscopy (STED, STORM, PALM): Overcomes diffraction limit for nanoscale localization

  • Electron microscopy with immunogold labeling: For ultrastructural localization

• Subcellular fractionation combined with immunoblotting:

  • Separate cellular components (membrane, cytosol, ER, Golgi)

  • Perform Western blot analysis on fractions using HTR3E antibody

  • Include markers for various compartments (e.g., calnexin for ER, GM130 for Golgi)

• Co-localization with organelle markers:

  • ER markers (calnexin, PDI): Particularly important as HTR3E is "presumably retained within the endoplasmic reticulum unless complexed with HTR3A"

  • Membrane markers (Na+/K+ ATPase)

  • Trafficking markers (Rab GTPases)

• Live-cell imaging approaches:

  • Fluorescently tagged HTR3E constructs

  • Antibody internalization assays using biotin-conjugated HTR3E antibodies

  • FRAP (Fluorescence Recovery After Photobleaching) to assess receptor mobility

Research has demonstrated membrane localization of 5-HT3 receptor subunits in colon tissues specifically in "cell bodies of myenteric neurons," providing anatomical context for functional studies .

How can researchers quantitatively analyze HTR3E expression patterns?

Quantitative analysis is essential for comparing expression levels across experimental conditions:

• Western blot quantification:

  • Densitometry analysis normalized to housekeeping proteins (GAPDH, β-actin)

  • Use of standard curves with recombinant HTR3E protein

  • Digital imaging systems such as "Odyssey Infrared Imaging system" provide accurate quantification with wide dynamic range

• Immunofluorescence quantification methods:

  • Mean fluorescence intensity measurements in defined regions of interest

  • Cell counting for percentage of positive cells in tissue sections

  • Colocalization coefficients (Pearson's, Manders') for assessing spatial relationships with other proteins

• Flow cytometry analysis:

  • Mean/median fluorescence intensity measurements

  • Percent positive cells relative to isotype controls

  • Multi-parameter analysis to correlate HTR3E expression with cell type markers

• RT-qPCR correlation:

  • Quantify HTR3E mRNA levels using appropriate primers and probes

  • Correlate transcript levels with protein expression

  • Analyze relationships between HTR3E and other 5-HT3 receptor subunits

A comprehensive approach might include both protein and mRNA quantification as demonstrated in studies examining miRNA regulation where "high miR-539 levels coincided with low HCS mRNA levels" showing reciprocal relationship between regulatory factors and their targets .

What are common challenges and solutions when working with biotin-conjugated HTR3E antibodies?

Researchers frequently encounter specific technical challenges that can be addressed through systematic troubleshooting:

Implementation of proper controls is essential for distinguishing technical issues from biological findings. As noted in research protocols, "specific signal was abolished after preadsorption with 100 μg of the immunogen," providing a valuable control for verifying antibody specificity .

How does biotin conjugation affect HTR3E antibody performance across different applications?

Biotin conjugation can influence antibody performance in several important ways:

• Epitope recognition impacts:

  • If biotin conjugation occurs near the antigen-binding site, it may reduce affinity

  • Conformational changes induced by conjugation may alter binding characteristics

  • Multiple biotin molecules can affect antibody stereochemistry and binding kinetics

• Application-specific considerations:

  • ELISA: Biotin conjugation generally enhances performance through improved signal amplification with streptavidin-HRP systems

  • Flow cytometry: May allow for flexible secondary detection but can increase background if endogenous biotin is present

  • Immunohistochemistry: Provides amplification benefits but requires careful blocking of endogenous biotin

• Optimization strategies:

  • Control the biotin-to-antibody ratio during conjugation (5:1 biotinylating reagent to antibody is standard)

  • Purify conjugated antibodies to remove free biotin (protein A affinity chromatography is effective)

  • Validate each batch of biotin-conjugated antibody against non-conjugated version to assess functional impacts

• Storage and stability considerations:

  • Biotin-conjugated antibodies should be stored at -20°C or -80°C

  • Addition of stabilizers (50% glycerol, BSA) helps maintain activity

  • Avoid repeated freeze-thaw cycles which particularly affect conjugated antibodies

What quality control parameters ensure reliable results with HTR3E antibodies?

Implementing rigorous quality control measures ensures experimental reliability:

• Pre-experimental validation:

  • Western blot analysis to confirm band at expected molecular weight (~51kDa for HTR3E)

  • Testing on positive control samples (tissues known to express HTR3E such as colon)

  • Verification of immunogen sequence specificity against related proteins

• Batch testing parameters:

  • Antibody concentration determination (absorbance at 280nm)

  • Biotin incorporation ratio assessment (HABA assay)

  • Functional activity testing in standard application (ELISA)

• Documentation requirements:

  • Antibody source, catalog number, lot number

  • Detailed experimental conditions including dilutions, incubation times/temperatures

  • Complete description of all controls employed

• Verification through multiple approaches:

  • Complementary techniques (Western blot, immunofluorescence, qPCR)

  • Multiple antibodies targeting different epitopes

  • Correlation with functional assays where applicable

Research publications emphasize validation through multiple methods, including "Western blot experiments by detecting bands of the expected sizes" combined with immunofluorescence showing "distinct patterns of immunofluorescence at expected sites" with confirmation through peptide blocking experiments .

How can HTR3E antibodies contribute to understanding serotonin receptor heterogeneity?

HTR3E antibodies provide valuable tools for exploring the complex landscape of serotonin receptor composition and function:

• Receptor subunit stoichiometry analysis:

  • Quantitative immunoprecipitation to determine relative abundance of different 5-HT3 subunits

  • Proximity ligation assays to verify physical interactions between HTR3E and other subunits

  • Super-resolution imaging to visualize subunit organization within receptor complexes

• Tissue-specific receptor profiles:

  • Comprehensive mapping of HTR3E distribution across tissues, particularly within the gastrointestinal tract

  • Correlation with functional responses to serotonergic drugs

  • Identification of tissue-specific interacting partners

• Pathophysiological significance:

  • Investigation of altered HTR3E expression in gastrointestinal disorders

  • Analysis of pharmacological responses in tissues with different subunit compositions

  • Potential development of subunit-selective therapeutic approaches

Research has already demonstrated that "HTR3E along with HTR3C and HTR3D cannot form functional 5-HT3 receptors on their own but require co-expression with HTR3A," revealing the importance of heteromeric assembly for receptor function . Future studies using biotin-conjugated HTR3E antibodies could further elucidate how these subunit combinations affect receptor pharmacology and signaling.

What emerging technologies might enhance HTR3E detection and analysis?

Several cutting-edge technologies offer new possibilities for HTR3E research:

• Advanced imaging approaches:

  • Expansion microscopy: Physical enlargement of specimens for super-resolution imaging with standard microscopes

  • Lattice light-sheet microscopy: For high-speed 3D imaging with reduced phototoxicity

  • Correlative light and electron microscopy (CLEM): Combining molecular specificity with ultrastructural context

• Single-cell analysis methods:

  • Single-cell proteomics to quantify HTR3E expression in individual cells

  • Spatial transcriptomics to correlate HTR3E protein localization with gene expression patterns

  • Mass cytometry (CyTOF) for high-dimensional protein profiling including HTR3E

• Innovative antibody engineering:

  • Site-specific conjugation strategies for precise biotin placement

  • Nanobodies/single-domain antibodies for improved tissue penetration

  • Click chemistry approaches for modular antibody functionalization

• Computational approaches:

  • Machine learning algorithms for automated image analysis of HTR3E staining patterns

  • Predictive modeling of receptor assembly based on subunit expression data

  • Integration of multi-omics data to contextualize HTR3E function

The "universal CAR T cells targeted to HER2 with a biotin-trastuzumab conjugate" approach demonstrates how biotin-conjugated antibodies can be leveraged in innovative therapeutic strategies, suggesting similar principles might apply to HTR3E research .

How might HTR3E antibody research contribute to therapeutic development?

HTR3E antibody-based research has potential therapeutic implications:

• Precision medicine applications:

  • Identification of patient subgroups with distinctive HTR3E expression patterns

  • Correlation of receptor subunit composition with response to 5-HT3 antagonists

  • Development of diagnostic tests to guide treatment selection

• Novel therapeutic approaches:

  • Targeted delivery of drugs to tissues expressing specific 5-HT3 receptor subtypes

  • Development of subunit-selective modulators based on structural insights

  • Antibody-drug conjugates directed at extracellular domains of HTR3E

• Biomarker development:

  • HTR3E expression as a potential prognostic or predictive marker

  • Correlation with treatment response to serotonergic agents

  • Monitoring receptor expression changes during disease progression

• Research translation requirements:

  • Validation in diverse patient populations

  • Standardization of detection methods for clinical implementation

  • Integration with existing diagnostic platforms