RORB Antibody, FITC conjugated

Introduction to RORB Antibody, FITC Conjugated

RORB Antibody, FITC conjugated is a specialized immunological reagent consisting of an antibody directed against the nuclear receptor ROR-beta (RORB) protein, chemically linked to fluorescein isothiocyanate (FITC), a commonly used fluorescent dye . The conjugation of FITC to the RORB antibody enables visualization of RORB protein expression in cells and tissues through fluorescence microscopy and flow cytometry applications.

RORB, also known as Nuclear receptor RZR-beta or Nuclear receptor subfamily 1 group F member 2 (NR1F2), is a nuclear receptor that functions as a transcription factor . It binds DNA as a monomer to ROR response elements (RORE) containing a single core motif half-site 5'-AGGTCA-3' preceded by a short A-T-rich sequence . RORB possesses intrinsic transcriptional activity and interacts with natural ligands such as all-trans retinoic acid (ATRA) and other retinoids, which act as inverse agonists repressing its transcriptional activity .

RORB Component

The RORB protein targeted by this antibody is a nuclear receptor with a molecular weight of approximately 52 kDa (459 amino acids) . RORB contains several functional domains including:

• DNA-binding domain responsible for recognizing specific DNA sequences

• Ligand-binding domain that interacts with regulatory molecules

• Hinge region connecting these domains

Multiple isoforms of RORB exist, with Rorb1 and Rorb2 being the most well-characterized . These isoforms show distinct expression patterns in different tissues, suggesting specialized roles in various biological processes.

FITC Component

Fluorescein isothiocyanate (FITC) is a derivative of fluorescein functionalized with an isothiocyanate reactive group (-N=C=S) . This modification enables FITC to form stable covalent bonds with primary amines and sulfhydryl groups on proteins, including antibodies . Key properties of FITC include:

• Excitation wavelength: 495 nm

• Emission wavelength: 519 nm (green fluorescence)

• Negatively charged ionic structure

• Susceptibility to photobleaching under prolonged exposure to excitation light

Applications in Research

RORB Antibody, FITC conjugated serves multiple research applications, primarily in visualizing and quantifying RORB expression in various cellular contexts.

Immunofluorescence Applications

This conjugated antibody is particularly valuable for immunofluorescence microscopy, allowing direct visualization of RORB protein localization without requiring secondary antibody detection steps . This application is especially useful for:

• Examining nuclear localization of RORB in tissue sections

• Co-localization studies with other proteins

• Evaluating RORB expression during development or under different experimental conditions

Flow Cytometry

FITC-conjugated antibodies can be employed in flow cytometry to quantify RORB expression in cell populations . This technique allows researchers to:

• Separate and analyze RORB-expressing cells

• Quantify expression levels across different cell populations

• Perform multiparameter analysis when combined with other fluorescent markers

ELISA and Other Immunoassays

Both commercially available RORB Antibody, FITC conjugated products are validated for ELISA applications , enabling quantitative analysis of RORB protein levels in various samples.

RORB Biology and Research Implications

Understanding RORB function provides context for applications of RORB Antibody, FITC conjugated in research studies.

Developmental Neurobiology

RORB plays critical roles in neural development, particularly in:

• Normal postnatal development of rod and cone photoreceptor cells

• Modulation of rod photoreceptor differentiation through NRL-mediated pathways

• Regulation of transcription factors in cone photoreceptor cells

• Control of cytoarchitectural patterning of neocortical neurons

Circadian Rhythm Regulation

RORB is involved in the regulation of period length and stability of circadian rhythms , making the FITC-conjugated antibody valuable for chronobiology research.

Articular Protection and Musculoskeletal Biology

Recent research has revealed that RORB modulates a gene program that is protective against articular degradation, with specific effects including:

• Reduction of the FGFR1/FGFR3 ratio toward a protective profile

• Alteration of phosphorylation patterns of ERK1 (decreased) and AKT (increased)

• Protection against IL-1β mediated inflammation

• Inhibition of basal protease production, including MMPs and ADAMTS4

Technical Considerations for Use

Several important considerations should be addressed when using RORB Antibody, FITC conjugated:

Photobleaching Prevention

FITC-conjugated antibodies are susceptible to photobleaching, especially under UV+blue light excitation . Researchers should:

• Minimize exposure to excitation light

• Store conjugates protected from light

• Consider anti-fade mounting media for preserved samples

• Be aware that FITC fluorescence contains components that fade with different kinetics

Background Fluorescence Management

FITC is an ionic fluorochrome (negatively charged) which can bind strongly to positively charged proteins, potentially creating background issues . Strategies to mitigate this include:

• Using appropriate blocking reagents (human IgG at 2% concentration has shown effectiveness)

• Considering alternative neutral fluorochromes like BODIPY FL for tissues with high positive charge content

• Implementing proper chromatography techniques, such as DEAE-cellulose chromatography, which can improve staining contrast

Optimization of Antibody Concentration

For optimal results, antibody concentrations of 0.5-5 mg/ml are recommended when working with FITC conjugation kits . The specific dilution factor will depend on the particular application and sample type.

Comparison with Alternative Conjugated Antibodies

When selecting a fluorescently labeled antibody for RORB detection, researchers should consider the relative merits of different conjugates:

Table 2: Comparison of FITC vs. Alternative Antibody Conjugates

Studies comparing FITC and lissamine rhodamine (RB 200) have found that after appropriate chromatography, rhodamine conjugates can provide better contrast and more resistance to photobleaching than FITC conjugates .

Future Research Directions

The development and application of RORB Antibody, FITC conjugated continues to evolve, with several promising directions:

1. Development of conjugates with improved photostability for long-term imaging experiments

2. Exploration of RORB function in additional tissue contexts, particularly in musculoskeletal and neurodevelopmental disorders

3. Application of this antibody in high-throughput screening for compounds that modulate RORB activity

4. Integration with newer technologies such as multiplexed imaging and spatial transcriptomics

The continued refinement of RORB Antibody, FITC conjugated reagents and protocols will likely enable more sophisticated investigations into RORB biology and its implications for development, circadian rhythm regulation, and disease processes.