OPN3 Antibody, HRP conjugated

What is OPN3 and why are OPN3 antibodies important in research?

OPN3 (Opsin 3) is a member of the G protein-coupled receptor superfamily classified as a photoreceptive non-visual opsin. It is strongly expressed in brain and testis, with weaker expression detected in liver, placenta, heart, lung, skeletal muscle, kidney, and pancreas . OPN3 antibodies are essential tools for characterizing protein expression patterns, subcellular localization, and potential functions in various tissues. The development of specific antibodies has been particularly crucial for neuroscience research, where traditional methods have faced challenges in detecting this protein in complex neural tissues .

How does HRP conjugation enhance the utility of OPN3 antibodies?

HRP conjugation provides distinct advantages for OPN3 detection:

This conjugation approach is particularly valuable for detecting OPN3, which may be expressed at relatively low levels in certain tissues . The HRP enzyme catalyzes reactions with substrates to generate detectable signals, making it ideal for applications requiring high sensitivity.

What tissue types express OPN3 and can be targeted with OPN3 antibodies?

Based on the literature, OPN3 shows distinct expression patterns across multiple tissues:

• Brain: Particularly in distinct layers of the cerebral cortex, hippocampal formation, and thalamic nuclei

• Testis: Strong expression reported

• Other tissues: Liver, placenta, heart, lung, skeletal muscle, kidney, and pancreas (with weaker expression)

• Cell types: Both neuronal (including GABAergic neurons) and non-neuronal cells

The OPN3-mCherry knock-in mouse model has revealed previously undocumented brain subregions expressing OPN3, highlighting how antibody-based approaches can validate and expand upon these findings in different species and experimental contexts .

How can different epitope-targeting OPN3 antibodies provide complementary data?

Multiple antibodies targeting different regions of OPN3 can provide more comprehensive insights:

Using antibodies targeting different epitopes provides complementary information about protein conformation, processing, and interactions. For instance, if a C-terminal antibody fails to detect OPN3 while a middle-region antibody shows positive staining, this might indicate C-terminal processing or masking due to protein-protein interactions .

How do OPN3 expression patterns in the brain compare between antibody-based detection and reporter gene models?

The development of the OPN3-mCherry knock-in mouse model has revealed important insights:

• The model was specifically created to overcome limitations of antibody-based detection, suggesting challenges with traditional immunodetection methods for OPN3

• This genetic approach revealed OPN3 expression in:

  • Previously undocumented brain subregions

  • GABAergic neurons

  • Non-neuronal cells

  • Punctate subcellular localization in the soma

• The reporter model provides advantages for developmental studies as OPN3 has been shown to be developmentally regulated in the brain

• The fusion protein approach allows for native promoter control and endogenous expression levels, providing a more physiologically relevant view than conventional antibody-based approaches

This comparison highlights how complementary approaches (antibody-based and genetic reporters) can provide a more complete understanding of OPN3 biology.

What are the optimal working dilutions for OPN3-HRP antibodies in different applications?

Based on the available data, optimal dilutions vary by application and specific antibody:

For HRP-conjugated antibodies specifically (like ABIN7162242), manufacturers note that "optimal working dilution should be determined by the investigator" . This highlights the importance of titration experiments to determine the optimal signal-to-noise ratio for each specific experimental context.

What controls are essential when validating OPN3-HRP antibody specificity?

Essential controls for validating OPN3-HRP antibody specificity include:

• Genetic controls: Tissues from OPN3 knockout or knockdown models represent the gold standard negative control. The OPN3-mCherry knock-in mouse could serve as both a positive control and reference for expression patterns

• Peptide competition: Pre-incubation of the antibody with the immunizing peptide (described as "a synthetic peptide corresponding to a sequence within amino acids 300-400 of human OPN3") should abolish specific staining

• Cross-validation: Compare results with multiple antibodies targeting different OPN3 epitopes (AA 313-402, AA 300-400, AA 161-210)

• Molecular weight verification: Western blots should detect bands at the expected molecular weight (listed as "35kDa/44kDa"), with the variation potentially reflecting glycosylation or other post-translational modifications

How can researchers minimize non-specific binding when using OPN3-HRP antibodies?

To optimize specificity when working with OPN3-HRP antibodies:

• Buffer composition: Consider the preservation buffers used for commercial antibodies:

  • "50% Glycerol, 0.01M PBS, pH 7.4" with "0.03% Proclin 300" preservative

  • "PBS with 0.02% sodium azide, 50% glycerol, pH 7.3"

• Antibody selection: Choose antibodies with demonstrated specificity. Several OPN3 antibodies are described as ">95%, Protein G purified" or "affinity purification"

• Washing protocols: Increase wash duration or detergent concentration (Tween-20) to reduce background without compromising specific signals

• Antibody titration: As indicated in the technical information, use recommended dilutions (1:500-1:2000 for Western blot) and optimize through titration experiments

• Blocking optimization: Use appropriate blocking solutions (typically 5% BSA or milk in TBST) matched to the antibody type

What factors affect cross-reactivity of OPN3 antibodies across different species?

The search results indicate varying cross-reactivity profiles:

When interpreting variable cross-reactivity:

• Sequence homology: OPN3 sequence conservation at epitope regions determines cross-reactivity potential. Different regions may have different degrees of conservation across species.

• Epitope-specific considerations: Antibodies targeting different regions (AA 313-402, AA 300-400, AA 161-210) will show different cross-reactivity patterns based on conservation of these specific sequences

• Validation requirements: Species-specific validation is essential, particularly for studies examining subtle differences in expression patterns or levels

How can discrepancies between OPN3 antibody labeling and other expression data be reconciled?

Discrepancies between OPN3 protein detection by antibodies and other methods (mRNA expression, reporter models) may arise from several factors:

• Technical sensitivity differences: Detection methods for mRNA (e.g., in situ hybridization, RT-PCR) and protein (immunohistochemistry, Western blotting) have different sensitivity thresholds

• Developmental regulation: The search results note that "OPN3 has been shown to be developmentally regulated, at least in the brain" . Temporal dynamics may result in mRNA expression preceding detectable protein accumulation

• Antibody limitations: As acknowledged in the research, "there had been a significant lack of encephalic opsin 3 (OPN3) protein characterization, likely driven by the absence of murine OPN3 antibodies"

• Post-translational regulation: OPN3 may undergo modifications that affect antibody recognition but not genetic reporter detection

The OPN3-mCherry knock-in mouse model offers a valuable tool for resolving such discrepancies by allowing direct visualization of the OPN3 protein under native promoter control .

What approaches can quantify OPN3 expression levels using HRP-conjugated antibodies?

Several quantitative methods are appropriate for analyzing OPN3 expression:

• Western blot densitometry: Semi-quantitative analysis of band intensity normalized to loading controls. Appropriate for comparing OPN3 levels across samples or conditions.

• ELISA quantification: Several OPN3 antibodies are validated for ELISA applications, allowing standard curve-based quantification for absolute protein determination

• Immunohistochemistry image analysis:

  • Cell counting: Percentage of OPN3-positive cells in defined regions

  • Optical density measurements: For DAB-based detection systems

  • Threshold-based area measurements: Percentage of tissue area showing OPN3 immunoreactivity

• Subcellular quantification: Given the "punctate subcellular localization in the soma" described for OPN3, quantifying puncta size, number, and intensity may provide insights into OPN3 regulation

How can researchers correlate OPN3 expression with functional outcomes in neuronal studies?

To establish meaningful correlations between OPN3 expression and function:

• Cell type-specific analysis: The research indicates OPN3 expression in "GABAergic neurons and non-neuronal cells" . Co-labeling strategies can correlate OPN3 expression with functional markers of specific neuronal subtypes.

• Circuit-level analysis: The detailed expression map in "distinct layers of the cerebral cortex, the hippocampal formation, distinct nuclei of the thalamus" provides anatomical context for circuit-level functional studies

• Light sensitivity assays: As a non-visual opsin, OPN3 may mediate light responses. Photostimulation protocols can be correlated with OPN3 expression patterns.

• Comparative approaches: The OPN3-mCherry knock-in mouse model enables direct comparison between:

  • Wild-type tissues with endogenous OPN3

  • OPN3 knockout models

  • The OPN3-mCherry knock-in model

• Developmental correlation: Given that "OPN3 has been shown to be developmentally regulated," researchers can correlate developmental expression patterns with the emergence of specific functional properties

What are promising research applications for OPN3-HRP antibodies beyond current uses?

Emerging research directions for OPN3-HRP antibodies include:

• Extracellular vesicle (EV) studies: Detecting OPN3 in EVs could reveal new intercellular signaling mechanisms

• Live tissue imaging: Developing cell-permeable HRP substrates compatible with OPN3-HRP antibodies for dynamic studies

• Proximity labeling: Using HRP-conjugated OPN3 antibodies for proximity-dependent biotinylation to identify OPN3 interaction partners

• Pathology correlations: Examining OPN3 expression changes in neurological disorders, potentially as a biomarker

• Comparative physiology: The varying species reactivity profiles of different OPN3 antibodies enable evolutionary studies across species

What technological advances might improve OPN3 detection methodologies?

Several technological advances could enhance OPN3 detection in the near future:

• Single-molecule detection systems that reduce the required amount of antibody while increasing sensitivity

• Multiplexed detection platforms combining OPN3-HRP antibodies with other cellular markers for comprehensive phenotyping

• Microfluidic-based automated immunoassays for high-throughput screening of OPN3 expression in patient samples or model systems

• Advanced tissue clearing methods compatible with HRP-based detection to enable whole-organ imaging of OPN3 expression patterns

• Computational approaches for integrating antibody-based detection with data from genetic reporters like the OPN3-mCherry model to build comprehensive expression atlases