OPN3 Antibody, Biotin conjugated

What is OPN3 and why are biotin-conjugated antibodies valuable for its detection?

OPN3 (Opsin 3, also known as encephalopsin or panopsin) is a G-protein coupled receptor (GPCR) belonging to the opsin family. In humans, the canonical OPN3 protein comprises 402 amino acid residues with a molecular mass of approximately 44.9 kDa . OPN3 functions as a photoreceptor in non-visual light perception pathways and is expressed in various tissues including the brain, skin, and retina .

Biotin-conjugated OPN3 antibodies offer significant advantages in research applications:

• Enhanced sensitivity: The biotin-streptavidin system provides signal amplification, enabling detection of low-abundance OPN3 protein

• Versatility: Compatible with multiple detection systems using streptavidin conjugated to various reporter molecules (fluorophores, enzymes)

• Stability: Biotin conjugation typically maintains antibody specificity while improving shelf-life

• Reduced background: Particularly valuable in tissues with high autofluorescence

Available biotin-conjugated OPN3 antibodies include polyclonal antibodies targeting specific regions such as amino acids 313-402 of human OPN3 .

What experimental applications are appropriate for biotin-conjugated OPN3 antibodies?

Biotin-conjugated OPN3 antibodies have been validated for several research applications:

For optimal results, researchers should:

• Perform antibody titration experiments

• Include appropriate positive controls (tissues with known OPN3 expression)

• Use blocking peptides to confirm specificity

• Consider tissue-specific optimization, as OPN3 expression varies significantly between tissues

How can researchers validate the specificity of biotin-conjugated OPN3 antibodies?

Establishing antibody specificity is critical for reliable experimental outcomes. Recommended validation approaches include:

• Blocking peptide experiments: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining, as demonstrated with the Opsin 3 (extracellular) Blocking Peptide (BLP-OR023)

• Western blot analysis: Verify a single band at the expected molecular weight (35-44 kDa for OPN3)

• Comparative analysis across tissues: Test antibody performance in tissues with known differential expression of OPN3

• Genetic models: Compare staining between wild-type tissues and those from OPN3 knockout models or using OPN3-mCherry knock-in mice

• Orthogonal validation: Compare protein detection with mRNA expression patterns using in situ hybridization

Example validation data from literature: "Western blot analysis of rat brain membranes and mouse brain lysate showed specific OPN3 detection that was abolished with blocking peptide pre-incubation" .

How can researchers use biotin-conjugated OPN3 antibodies to investigate OPN3's expression in the nervous system?

OPN3's expression in neural tissues requires sophisticated experimental approaches:

• Co-localization studies: Combine biotin-conjugated OPN3 antibodies with markers for specific neural populations:

  • For example, in the hippocampus, OPN3 immunoreactivity has been demonstrated in the CA3 region, specifically in the pyramidal layer and mossy fiber terminal zone

  • In the PVN (paraventricular nucleus), OPN3 co-localizes with MC4R-expressing neurons

• Subcellular localization: High-resolution imaging reveals:

  • OPN3 displays punctate subcellular localization in neuronal soma

  • OPN3-mCherry knock-in mice show expression in distinct layers of the cerebral cortex and hippocampal formation

• Regional distribution mapping: Systematic analysis shows:

  • OPN3 is expressed in "distinct layers of the cerebral cortex (CTX), the hippocampal formation (HCF), distinct nuclei of the thalamus, as well as many other regions in both neuronal and non-neuronal cells"

  • In the hypothalamus, OPN3 is found in both anterior and posterior portions of the PVN and periventricular nuclei

Recommended methodological approach: "Immunohistochemical staining of perfusion-fixed frozen mouse brain sections with Anti-Opsin 3 (extracellular) Antibody (#AOR-023), (1:300), followed by goat anti-rabbit-AlexaFluor-488" .

What strategies should researchers employ to investigate OPN3's role in melanocyte biology?

OPN3 plays a significant role in melanocyte function, particularly in pigmentation regulation:

• Functional interactions with melanocortin receptors:

  • OPN3 "acts as a negative regulator of melanin production by modulating the signaling of MC1R"

  • "OPN3 negatively regulates the cyclic adenosine monophosphate (cAMP) response evoked by MC1R via activation of the Gαi subunit of G proteins"

• Experimental design for mechanistic studies:

  • Use biotin-conjugated OPN3 antibodies in co-immunoprecipitation experiments to detect OPN3-MC1R complexes

  • Combine with proximity ligation assays to confirm direct interaction in situ

  • Employ primary human epidermal melanocytes (HEMs) for physiologically relevant models

• Light-dependent signaling investigations:

  • Design experiments that control for wavelength-specific effects (blue light vs. UVA)

  • Use biotin-conjugated OPN3 antibodies for immunofluorescence before and after light exposure to detect potential translocation

• Downstream signaling analysis:

  • Monitor OPN3-dependent changes in calcium flux, CAMK2 phosphorylation, and CREB/p38/ERK/MITF phosphorylation

  • Combine antibody-based detection with functional readouts like melanin quantification

Research insight: "OPN3 and MC1R colocalize at both the plasma membrane and in intracellular structures, and can form a physical complex" .

How should researchers design experiments to distinguish between different OPN3 epitopes and isoforms?

OPN3 antibodies target different epitopes, requiring careful experimental design:

• Epitope mapping strategies:

  • Available antibodies target diverse regions of OPN3, including:

    • AA 161-210 (ABIN1535598)

    • AA 180-194 (mouse sequence, extracellular 2nd loop, AOR-023)

    • AA 300-400 (ABIN7269102)

    • AA 313-402 (recombinant human protein sequence)

• Isoform-specific detection:

  • Human OPN3 has at least 2 isoforms

  • Select antibodies recognizing unique regions specific to each isoform

  • Consider combining antibody-based detection with RT-PCR for isoform-specific transcripts

• Cross-reactivity assessment:

  • Test antibodies against recombinant OPN3 from different species

  • Perform comparative analysis in tissues with different OPN3 isoform expression patterns

• Validation with genetic tools:

  • Compare antibody signal between wild-type tissues and OPN3-mCherry knock-in mice

  • Use species-specific amino acid sequences as controls for antibody specificity

Technical consideration: "Anti-Opsin 3 (extracellular) Antibody targets the peptide (C)DIHGLG(S)TVDWRSKD, corresponding to amino acid residues 180-194 of mouse Opsin 3 (Accession Q9WUK7)" .

What methodological approaches are optimal for investigating OPN3 in insect models?

Research on OPN3 homologues in insect systems presents unique experimental challenges:

• In situ hybridization protocols:

  • "Preparation of RNA probes and in situ hybridization were carried out using DIG- and biotin-labelled antisense and sense RNA probes for A. stephensi MosOpn3 (Asop12)"

  • "DIG-labelled probes were visualized with an alkaline phosphatase-conjugated anti-DIG antibody, followed by a blue 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium color reaction"

• Tissue-specific expression analysis:

  • "The expression of MosOpn3 mRNA was specifically detected in ommatidia located in dorsal and ventral areas of the compound eye in the frontal section"

  • "MosOpn3 is specifically expressed in R7 in dorsal and ventral ommatidia in the mosquito"

• Co-expression studies with visual opsins:

  • Design double in situ hybridization experiments to distinguish between visual and non-visual opsins

  • Combine with immunohistochemistry using appropriate cross-reactive antibodies

• Comparative evolutionary approaches:

  • Select antibodies recognizing conserved epitopes when studying insect OPN3 homologues

  • Use phylogenetic analyses to inform experimental design and interpretation

Methodological insight: "In double in situ hybridization, DIG-labelled probes for MosOpn3 and biotin-labelled probes for Asop1, Asop8 or Asop9 were used" to distinguish expression patterns between different opsin types .

What are the most common technical challenges when using biotin-conjugated OPN3 antibodies?

Researchers should anticipate and address several technical challenges:

• Signal-to-noise ratio optimization:

  • Biotin-conjugated OPN3 antibodies typically require dilution optimization (ranges from 1:10 to 1:2000 depending on application)

  • Include proper blocking steps to minimize non-specific binding

  • Consider endogenous biotin blocking when working with biotin-rich tissues

• Tissue-specific considerations:

  • Brain tissues may require specialized fixation protocols to preserve OPN3 epitopes

  • For melanocytes, minimize melanin interference with detection systems

  • In retinal tissues, account for high autofluorescence

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize buffer conditions (citrate vs. EDTA-based buffers)

  • Determine optimal incubation times to maximize epitope exposure while preserving tissue morphology

• Detection system selection:

  • For fluorescence applications, choose fluorophores that minimize overlap with tissue autofluorescence

  • For chromogenic detection, optimize development times to achieve optimal signal-to-noise ratio

Example optimization protocol: "For Western blot analysis, use antibody dilutions of 1:500~1:1000; for immunofluorescence, use 1:10" .

How can researchers overcome challenges in detecting low-abundance OPN3 in specific tissues?

OPN3 detection in tissues with low expression levels requires specialized approaches:

• Signal amplification strategies:

  • Leverage the biotin-streptavidin system's amplification capabilities

  • Consider tyramide signal amplification (TSA): "Biotin-labelled probes were visualized with the TSA system (Perkin Elmer), followed by horseradish peroxidase diaminobenzidine reaction"

  • Use enhanced chemiluminescence detection for Western blots

• Sample enrichment techniques:

  • Consider membrane fraction isolation for Western blot applications

  • Use laser capture microdissection to isolate specific cell populations

  • Employ immunoprecipitation to concentrate OPN3 before analysis

• Sensitive detection methods:

  • Utilize confocal microscopy with appropriate spectral settings

  • Consider super-resolution microscopy for subcellular localization studies

  • Use cooled CCD cameras with extended exposure times for fluorescence imaging

• Controls and validation:

  • Include positive control tissues with known high OPN3 expression

  • Use recombinant OPN3 protein as positive control for Western blots

  • Compare results from multiple OPN3 antibodies targeting different epitopes

Research insight: "OPN3-mCherry was readily visualized in distinct layers of the cerebral cortex (CTX), the hippocampal formation (HCF), distinct nuclei of the thalamus, as well as many other regions in both neuronal and non-neuronal cells" .

What buffer and storage conditions best preserve biotin-conjugated OPN3 antibody activity?

Proper handling is critical for maintaining antibody performance:

• Recommended storage conditions:

  • "Aliquot and store in the dark at 2-8°C. Keep protected from prolonged exposure to light"

  • "Avoid repeated freeze/thaw cycles"

  • "Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use"

• Buffer composition considerations:

  • Common storage buffers include:

    • "PBS (pH 7.2)"

    • "0.01 M PBS, pH 7.4, 0.03% Proclin-300 and 50% glycerol"

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

• Reconstitution and dilution guidelines:

  • Follow manufacturer's instructions for reconstitution of lyophilized antibodies

  • Prepare working dilutions fresh before use

  • Use high-quality, protein-free diluents for optimal results

• Long-term preservation strategies:

  • For extended storage, maintain at -20°C in small aliquots

  • Include carrier proteins (BSA) in working dilutions to prevent adsorption to tube walls

  • Monitor antibody performance periodically with positive control samples

Technical note: "For laboratory research only, not for drug, diagnostic or other use" .

How can researchers leverage biotin-conjugated OPN3 antibodies to investigate light-dependent signaling in non-retinal tissues?

OPN3's role in light sensing beyond the retina represents an exciting research frontier:

• Experimental design for light-dependent studies:

  • "OPN3 selectively activates G proteins via ultraviolet A (UVA) light-mediated activation in the skin"

  • Design protocols that control light exposure (wavelength, intensity, duration)

  • Combine antibody detection with functional readouts (calcium imaging, cAMP measurement)

• Tissue-specific considerations:

  • For melanocytes: "OPN3 regulates melanogenesis via inhibition of alpha-MSH-induced MC1R-mediated cAMP signaling, modulation of calcium flux, regulation of CAMK2 phosphorylation, and subsequently phosphorylation of CREB, p38, ERK and MITF in response to blue light"

  • For dermal fibroblasts: Study "UVA-mediated induction of calcium and mitogen-activated protein kinase signaling resulting in the expression of MMP1, MMP2, MMP3, MMP9 and TIMP1"

  • For adipocytes: Investigate "light-mediated glucose uptake, mitochondrial respiration and fatty acid metabolism"

• Proteomic approaches:

  • Use biotin-conjugated OPN3 antibodies for pull-down experiments before and after light exposure

  • Identify differential protein interactions using mass spectrometry

  • Validate findings with co-immunoprecipitation and proximity ligation assays

• Translational research opportunities:

  • Investigate "photorelaxation of airway smooth muscle cells, via blue-light dependent GPCR signaling pathways"

  • Study keratinocyte differentiation in response to blue light

Research insight: "OPN3 binds both 11-cis retinal and all-trans retinal" and can undergo conformational changes upon light exposure .

What approaches should researchers use to investigate OPN3's interactions with G-protein signaling pathways?

Understanding OPN3's coupling to G-protein signaling requires sophisticated experimental strategies:

• G-protein coupling specificity:

  • "OPN3 negatively regulates the cAMP response evoked by MC1R via activation of the Gαi subunit of G proteins"

  • Design experiments with specific G-protein inhibitors (pertussis toxin for Gαi)

  • Use FRET/BRET-based assays to monitor G-protein activation in real-time

• Interaction with second messenger systems:

  • OPN3 affects calcium flux and cAMP levels in various cell types

  • Combine antibody-based detection with functional readouts (ELISA for cAMP, calcium imaging)

  • Use pharmacological approaches to dissect specific signaling components

• Cross-talk with other GPCRs:

  • "OPN3 suppresses MC4R signaling" in the hypothalamus

  • OPN3 can form complexes with MC1R in melanocytes

  • Design co-immunoprecipitation experiments using biotin-conjugated OPN3 antibodies

  • Perform proximity ligation assays to confirm interactions in situ

• Downstream effector analysis:

  • Study phosphorylation of key signaling molecules (CREB, p38, ERK)

  • Investigate effects on ion channel function (Kir7.1)

  • Use proteomics to identify novel downstream targets

Methodological insight: "OPN3 can form complexes with and regulate the activity of both MC4R and Kir7.1" indicating a novel regulatory mechanism for this non-visual opsin .

How can biotin-conjugated OPN3 antibodies contribute to understanding OPN3's role in metabolic regulation?

Recent research has revealed OPN3's unexpected role in metabolic processes:

• Hypothalamic control of food intake:

  • "Deletion of OPN3 in MC4R-expressing neurons results in reduced food consumption"

  • "OPN3 conditional heterozygotes and homozygotes show reduced food intake and locomotor activity"

  • Design immunohistochemistry experiments to map OPN3 expression in feeding-related neural circuits

• Energy homeostasis investigations:

  • "Opn3 null mice exhibit changes in metabolic homeostasis including reduced food consumption"

  • Combine antibody-based OPN3 detection with metabolic phenotyping (calorimetry, glucose tolerance)

  • Correlate OPN3 expression levels with metabolic parameters

• Tissue-specific knockout studies:

  • Use biotin-conjugated OPN3 antibodies to confirm successful deletion in conditional knockout models

  • Design tissue-specific deletion strategies targeting metabolically relevant tissues

  • Validate findings with comprehensive metabolic phenotyping

• Circadian regulation of metabolism:

  • Investigate OPN3's role in synchronizing metabolic processes with light cues

  • Design time-course experiments sampling across the circadian cycle

  • Correlate OPN3 expression/activity with metabolic rhythms

Research insight: "The deletion of Opn3 in Mc4r-expressing neurons results in reduced food intake, consistent with a physiological role for OPN3 in promoting food consumption" .