HTR7 Antibody, Biotin conjugated

What is HTR7 and why is it significant in neuroscience research?

HTR7 (5-hydroxytryptamine receptor 7) is a G protein-coupled receptor (GPCR) that functions as one of the serotonin receptor subtypes in the central nervous system (CNS). It plays important roles in neuromodulation and neurotransmission processes. HTR7 has a calculated molecular weight of approximately 49 kDa, though it is typically observed at 50-55 kDa in Western blot applications .

Significant research interest surrounds HTR7 due to its involvement in:

• Serotonergic signaling pathways in the brain

• Potential associations with mood disorders

• Response mechanisms to selective serotonin reuptake inhibitors (SSRIs)

Recent findings have identified functional variants in the HTR7 gene, such as rs7905446, which are associated with favorable responses to SSRIs in both bipolar and unipolar depression, suggesting HTR7's importance in antidepressant action mechanisms .

What are the primary applications for HTR7 Antibody, Biotin conjugated?

The biotin-conjugated HTR7 antibody is primarily utilized in the following applications:

The biotin conjugation specifically enhances detection sensitivity through biotin-streptavidin interaction systems. In ELISA applications, the biotin-conjugated antibody typically functions as a detection antibody in sandwich ELISA formats where it binds to the target HTR7 protein that has been captured by another antibody coated on the microwell surface .

How should HTR7 Antibody, Biotin conjugated be stored and handled?

Proper storage and handling are crucial for maintaining antibody performance:

• Storage temperature: Upon receipt, store at -20°C or -80°C

• Buffer composition: Typically preserved in 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4

• Avoid repeated freeze-thaw cycles as this can degrade antibody performance

• Form: Supplied as a liquid

• Shelf life: Stable for one year after shipment when stored properly

For optimal results, aliquot the antibody upon receipt to minimize freeze-thaw cycles. When handling, keep the antibody on ice during experimental setup and return to storage promptly after use.

How is the HTR7 Antibody, Biotin conjugated validated for specificity?

Validation of HTR7 antibody specificity involves multiple approaches:

• Immunogen verification: The antibody is typically raised against recombinant Human 5-hydroxytryptamine receptor 7 protein (amino acids 1-83) , which represents a specific epitope region.

• Species reactivity: Most commercially available HTR7 antibodies demonstrate reactivity with human samples, while some show cross-reactivity with mouse and rat samples .

• Application-specific validation:

  • For ELISA: Tested using recombinant proteins and validated human samples

  • For Western blot: Verification of protein band at expected molecular weight (50-55 kDa)

• Purification quality: Typically protein G purified to >95% purity

When selecting an HTR7 antibody for your research, review the validation data provided by the manufacturer, including Western blot images and ELISA standard curves if available.

What controls should be included when using HTR7 Antibody, Biotin conjugated?

When designing experiments with HTR7 Antibody, Biotin conjugated, include these essential controls:

• Positive control:

  • Human brain tissue samples (for IHC applications)

  • Mouse brain tissue (shows positive WB detection)

  • Mouse spleen tissue (shows positive WB detection)

• Negative controls:

  • Secondary antibody-only control (omit primary antibody)

  • Isotype control (rabbit IgG at equivalent concentration)

  • Tissues known to lack HTR7 expression

• Biotin-specific controls:

  • Biotinylated Horseradish Peroxidase (40kDa) can serve as a biotinylation control

  • Include a biotin blocking step in protocols where endogenous biotin might interfere

• Absorption control: Pre-incubation of the antibody with the immunizing peptide to confirm binding specificity

How can high-biotin sample interference be mitigated in immunoassays using biotin-conjugated HTR7 antibody?

Biotin interference is a significant consideration when using biotin-conjugated antibodies, as high biotin levels in samples can compete with the biotin-streptavidin detection system:

• Sample pretreatment:

  • Implement a sample dilution strategy to reduce biotin concentration

  • Consider using streptavidin microbeads to pre-absorb excess biotin from samples

• Assay modification:

  • Utilize alternative detection systems that do not rely on biotin-streptavidin interaction

  • In ELISA applications, test alternative antibody pairs where the detection antibody uses a different conjugate (e.g., HRP-direct conjugated)

• Quantification and adjustment:

  • Use the Pierce Biotin Quantitation Kit to measure biotin content in samples

  • Perform a biotin interference test with each sample batch by spiking known amounts of biotin

  • Implement a correction factor based on the measured biotin interference

• Documentation:

  • Record lot-to-lot variations in biotin content of antibody preparations

  • Document any sample pretreatment procedures to ensure experimental reproducibility

What are the technical considerations for optimizing HTR7 detection in ELISA assays?

Optimizing HTR7 detection in ELISA requires systematic protocol refinement:

• Antibody concentration optimization:

  • Perform checkerboard titration to determine optimal primary and detection antibody concentrations

  • For biotin-conjugated HTR7 antibody, typical starting dilutions vary by manufacturer

• Sandwich ELISA considerations:

  • When using a sandwich ELISA format, ensure capture and detection antibodies recognize different epitopes

  • Standard ELISA protocol involves coating wells with capture antibody, adding samples containing HTR7, followed by biotin-conjugated HTR7 antibody and then streptavidin-HRP

• Signal development:

  • For optimal sensitivity, use substrates like TMB (3,3′,5,5′ tetra-methylbenzidine)

  • Monitor the color development and optimize incubation time (typically 10 minutes at 37°C)

• Standard curve preparation:

  • Prepare HTR7 standards using recombinant protein

  • Ensure a range that spans from below the expected limit of detection to the upper range of expected sample concentrations

  • Use a 4-parameter logistic curve fit for data analysis

• Sample preparation:

  • For serum/plasma samples, consider dilution to minimize matrix effects

  • For tissue samples, optimize protein extraction buffer composition

How can HTR7 Antibody, Biotin conjugated be used to investigate serotonin receptor signaling in neuroscience?

For sophisticated neuroscience research applications, HTR7 antibody can be leveraged in multiple experimental paradigms:

• Co-localization studies:

  • Use biotin-conjugated HTR7 antibody in combination with other fluorescently-labeled antibodies against signaling molecules

  • Employ streptavidin conjugated with fluorophores for visualization

  • This approach allows investigation of HTR7's spatial relationship with other proteins in neuronal circuits

• Functional studies:

  • Combine HTR7 antibody detection with calcium imaging to correlate receptor expression with functional responses

  • Integrate antibody-based protein detection with electrophysiological recordings

• Genetic variation studies:

  • Use HTR7 antibody to investigate protein expression levels in samples with different HTR7 genetic variants

  • Particularly relevant for studying functional variants like rs7905446 which has been associated with SSRI response in depression

• Developmental neurobiology:

  • Track HTR7 expression across developmental stages using the antibody

  • Correlate expression patterns with the formation of serotonergic circuits

What methods can be used to quantify the biotin:protein ratio in HTR7 antibody preparations?

Determining the degree of biotinylation is critical for optimizing experimental protocols:

• HABA/Avidin-based methods:

  • The HABA (4′-hydroxyazobenzene-2-carboxylic acid)/Avidin assay measures biotin displacement

  • When biotin displaces HABA from avidin, absorbance at 500nm decreases proportionally

  • This change in absorbance can be used to calculate biotin concentration using the extinction coefficient of the HABA-avidin complex

• Mass spectrometry analysis:

  • Provides precise determination of biotin:protein ratio

  • Can identify specific biotinylation sites on the antibody molecule

  • Useful for advanced research requiring detailed characterization

• Functional assessment:

  • Compare signal intensity between different lots of biotin-conjugated antibody

  • Use titration experiments to determine optimal working concentration

  • Create standard curves with biotinylated HRP control to normalize between experiments

How can potential cross-reactivity with other serotonin receptors be identified and minimized?

Ensuring specificity among the closely related serotonin receptor family requires rigorous validation:

• Epitope analysis:

  • Review the immunogen sequence used for antibody generation (typically amino acids 1-83 of HTR7)

  • Compare this sequence with other serotonin receptor subtypes using bioinformatics tools to identify potential cross-reactive regions

• Experimental verification:

  • Test the antibody against cell lines expressing different serotonin receptor subtypes

  • Perform Western blot analysis on tissues with known differential expression of serotonin receptors

  • Use knockout/knockdown models where available to confirm specificity

• Absorption testing:

  • Pre-incubate the antibody with recombinant proteins of related serotonin receptors

  • If signal is reduced after pre-incubation with non-target proteins, cross-reactivity is likely

• Isoform considerations:

  • Some HTR7 antibodies recognize specific isoforms (e.g., isoform a, NP_000863.1)

  • Confirm which isoforms are relevant to your research question

What are the considerations when using HTR7 antibody in studies of SSRI response and serotonergic pathways?

When investigating the relationship between HTR7 and antidepressant response:

• Genetic-protein correlation:

  • Recent research has identified HTR7 gene variants associated with SSRI response

  • The variant rs7905446 (T/G) located in the promoter region shows significant association with response to paroxetine and other SSRIs

  • Use HTR7 antibody to measure protein expression levels in samples with different genotypes

• Pharmacological validation:

  • Combine antibody-based detection with pharmacological manipulation using HTR7 agonists/antagonists

  • Measure receptor expression changes in response to SSRI treatment

• Temporal considerations:

  • Design time-course experiments to capture dynamic changes in HTR7 expression

  • Use the antibody to track receptor internalization or trafficking following serotonergic stimulation

• Regional specificity:

  • When studying brain tissue, consider region-specific expression patterns

  • Optimize immunohistochemistry protocols for different brain regions, which may require different fixation and antigen retrieval methods

What are common issues encountered when using HTR7 Antibody, Biotin conjugated and how can they be resolved?

How can I validate that my HTR7 Antibody, Biotin conjugated maintains activity after storage?

To ensure antibody performance over time:

• Activity testing:

  • Run a standard ELISA using a consistent positive control sample

  • Compare results to initial testing data to identify any sensitivity loss

• Biotin functionality check:

  • Use HABA/Avidin assay to verify biotin accessibility

  • Compare binding efficiency to fresh standards

• Storage validation:

  • Test antibody performance after different storage durations

  • Create a validation schedule based on usage frequency

  • Document performance metrics for each lot over time

• Quality control measures:

  • Store small reference aliquots from each new lot

  • Use these as benchmarks when testing antibody performance over time

  • Implement a minimum signal-to-noise ratio threshold for acceptable performance