MTNR1B Antibody, FITC conjugated

What is MTNR1B and what cellular functions does it regulate?

MTNR1B (Melatonin Receptor 1B) is a high-affinity receptor for melatonin that plays crucial roles in regulating reproductive and circadian actions of melatonin. At the molecular level, MTNR1B functions as a G protein-coupled receptor whose activity is mediated by pertussis toxin-sensitive G proteins that inhibit adenylate cyclase activity . This inhibitory action on adenylate cyclase represents a key mechanism by which melatonin exerts its physiological effects on target tissues. The receptor is primarily localized in the cytoplasm of cells and mediates melatonin's effects on circadian rhythms, glucose metabolism, and reproductive functions . Understanding MTNR1B's physiological roles is essential for researchers investigating sleep disorders, metabolic diseases, and seasonal reproductive variations.

What are the excitation and emission wavelengths for the FITC conjugate, and how should imaging parameters be optimized?

The FITC conjugate attached to the MTNR1B antibody has an excitation maximum at 494nm and an emission maximum at 518nm . When designing multicolor immunofluorescence experiments, researchers should consider these spectral properties to avoid bleed-through or cross-excitation with other fluorophores. For optimal imaging, use filter sets designed for FITC/green fluorescence (typically excitation filter ~490nm, emission filter ~525nm). To maximize signal quality, researchers should optimize exposure times to prevent photobleaching while maintaining adequate signal intensity. Additionally, appropriate negative controls should be included to establish background fluorescence levels and confirm signal specificity.

What species reactivity can be expected with MTNR1B Antibody, FITC conjugated?

The MTNR1B Antibody, FITC conjugated demonstrates reactivity with human (Hu), mouse (Ms), and rat (Rt) samples . This cross-species reactivity makes it a versatile tool for comparative studies across different mammalian models. The antibody's ability to recognize MTNR1B across multiple species is based on the conservation of the antigenic epitope (amino acids 222-253) within the MTNR1B protein sequence. Researchers working with other species should conduct preliminary validation experiments to confirm cross-reactivity before proceeding with full-scale studies.

What is the recommended protocol for sample preparation when using MTNR1B Antibody, FITC conjugated in fixed tissues?

When preparing samples for MTNR1B detection using FITC-conjugated antibodies, proper fixation and permeabilization are critical for optimal results. Based on successful applications, formalin fixation followed by paraffin embedding has been validated for MTNR1B detection in various tissues including human retina, brain, and skin melanoma samples . For immunohistochemical analysis, deparaffinize sections, perform antigen retrieval (typically using citrate buffer pH 6.0), block endogenous peroxidase activity and non-specific binding sites, and then incubate with the MTNR1B antibody at the recommended dilution (1:50-1:200 for IF applications) . For cultured cells, 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization is generally effective. When working with the FITC-conjugated antibody, minimize exposure to light throughout the procedure to prevent photobleaching of the fluorophore.

How should MTNR1B Antibody, FITC conjugated be stored to maintain optimal activity?

The MTNR1B Antibody, FITC conjugated should be shipped at 4°C and stored at -20°C for long-term preservation . The antibody is formulated in 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol to maintain stability . To preserve antibody activity, repeated freeze/thaw cycles should be avoided as they can lead to protein denaturation and loss of binding capacity. Aliquoting the antibody into smaller volumes upon receipt is recommended to minimize freeze-thaw cycles. When handling the antibody, always keep it on ice and protected from light due to the photosensitivity of the FITC fluorophore. Under optimal storage conditions, the antibody can maintain activity for approximately one year .

What controls should be included when using MTNR1B Antibody, FITC conjugated in research applications?

For rigorous experimental design, several controls are essential when using MTNR1B Antibody, FITC conjugated:

• Positive controls: Tissues or cells known to express MTNR1B, such as human retina, brain, or melanoma tissues that have been validated in previous studies .

• Negative controls: Include:

  • Primary antibody omission control (all reagents except the MTNR1B antibody)

  • Isotype control (non-specific IgG from same species at equivalent concentration)

  • Tissues/cells known not to express MTNR1B

• Blocking peptide control: Pre-incubation of the antibody with the immunizing peptide (synthetic peptide aa 222-253) to confirm binding specificity .

• Fluorescence controls: Auto-fluorescence control (unstained sample) and single-color controls when performing multi-color immunofluorescence to establish compensation settings.

These controls allow researchers to validate antibody specificity and distinguish true signal from technical artifacts.

How can MTNR1B Antibody, FITC conjugated be utilized in studying the relationship between MTNR1B polymorphisms and metabolic disorders?

MTNR1B gene polymorphisms, particularly the rs10830963 C/G variant, have been strongly associated with type 2 diabetes mellitus (T2DM) risk through mechanisms affecting insulin secretion and glucose metabolism . Researchers can employ MTNR1B Antibody, FITC conjugated to investigate how these polymorphisms affect receptor expression, localization, and signaling in cellular models. This approach can be particularly valuable in experiments comparing receptor dynamics between tissues from subjects with different genotypes.

A comprehensive experimental design might include:

• Genotyping samples for the rs10830963 C/G polymorphism

• Quantifying MTNR1B expression levels using the FITC-conjugated antibody in immunofluorescence or flow cytometry

• Correlating expression patterns with genotype and clinical parameters

• Examining co-localization with downstream signaling molecules

Such studies can provide mechanistic insights into how the risk allele G leads to MTNR1B overexpression in pancreatic islet cells, potentially contributing to decreased insulin secretion and elevated fasting blood glucose levels .

What methodological approaches can be used to investigate MTNR1B-mediated G protein signaling using the FITC-conjugated antibody?

MTNR1B signals through pertussis toxin-sensitive G proteins that inhibit adenylate cyclase activity . To investigate this signaling pathway, researchers can combine MTNR1B Antibody, FITC conjugated with complementary techniques:

• Co-localization studies: Use MTNR1B Antibody, FITC conjugated alongside antibodies against G protein subunits (labeled with spectrally distinct fluorophores) to visualize receptor-G protein interactions following melatonin stimulation.

• FRET-based approaches: Modify protocols to use the FITC-labeled MTNR1B antibody as a donor fluorophore in Förster resonance energy transfer experiments to detect molecular proximity with downstream signaling components.

• Time-course experiments: Apply the FITC-conjugated antibody to track receptor internalization and recycling following agonist stimulation.

• Signaling inhibitor studies: Combine antibody labeling with pertussis toxin treatments to confirm G protein involvement and correlate receptor localization with functional outcomes.

These approaches can help elucidate how MTNR1B mediates the inhibition of adenylate cyclase, which affects cyclic AMP levels and ultimately influences the action of incretin hormones like GLP-1 and GIP that regulate insulin secretion .

What considerations should be taken into account when using MTNR1B Antibody, FITC conjugated in multi-color immunofluorescence experiments?

When designing multi-color immunofluorescence experiments incorporating MTNR1B Antibody, FITC conjugated, several technical considerations are essential:

• Spectral overlap management: FITC (excitation: 494nm, emission: 518nm) has potential spectral overlap with other green-yellow fluorophores. Select companion fluorophores with minimal spectral overlap, such as DAPI (blue), Cy3 (red), or Cy5 (far-red).

• Sequential imaging: Consider acquiring images sequentially rather than simultaneously to minimize bleed-through, particularly if using fluorophores with close spectral properties.

• Antibody compatibility: When co-staining, ensure other primary antibodies are from different host species than the MTNR1B antibody (rabbit) to avoid cross-reactivity of secondary antibodies.

• Order of application: For multi-labeling protocols, determine the optimal order of antibody application, generally starting with the weakest signal (which may require the FITC-conjugated antibody to be applied first or last depending on target abundance).

• Cross-blocking: Implement appropriate blocking steps between different antibody applications to prevent non-specific binding.

A systematic approach to these considerations will enable successful co-localization studies examining MTNR1B expression in relation to other cellular components involved in melatonin signaling and glucose metabolism pathways.

What strategies can be employed to improve signal-to-noise ratio when using MTNR1B Antibody, FITC conjugated?

When working with MTNR1B Antibody, FITC conjugated, researchers may encounter challenges with signal-to-noise ratios. Several strategies can address these issues:

• Optimization of blocking conditions: Use a combination of 5-10% normal serum (from a species different from the host of primary antibody) with 1-3% BSA to reduce non-specific binding .

• Antibody titration: Systematically test dilutions within and beyond the recommended 1:50-1:200 range to identify the optimal concentration for specific samples .

• Autofluorescence reduction: Treat samples with sodium borohydride (1mg/ml for 10 minutes) or commercial autofluorescence quenchers, particularly important for tissues with high natural fluorescence like brain sections .

• Extended washing steps: Implement longer or additional washing steps (using 0.1% Tween-20 in PBS) to remove unbound antibody more effectively.

• Confocal microscopy settings: Adjust pinhole settings, gain, and laser power to optimize signal detection while minimizing background.

• Sample-specific fixation optimization: Different tissues may require modified fixation protocols; for example, shorter fixation times for better epitope preservation in sensitive tissues.

These approaches should be systematically tested and documented to establish optimal conditions for specific experimental systems.

How can researchers validate the specificity of MTNR1B Antibody, FITC conjugated in their experimental models?

Validating antibody specificity is critical for ensuring reliable and reproducible research results. For MTNR1B Antibody, FITC conjugated, consider these validation approaches:

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (synthetic peptide corresponding to amino acids 222-253 of human MTNR1B) before application to samples. Specific binding should be significantly reduced or eliminated.

• Knockout/knockdown controls: Compare staining between wild-type samples and those with MTNR1B knocked out (CRISPR/Cas9) or knocked down (siRNA), expecting reduced or absent signal in the latter.

• Correlation with mRNA expression: Perform parallel RT-PCR or in situ hybridization to correlate protein detection with mRNA expression patterns.

• Western blot validation: Confirm antibody specificity by Western blot analysis, looking for bands of appropriate molecular weight (approximately 40-45 kDa for MTNR1B).

• Cross-validation with independent antibodies: Compare staining patterns with alternative MTNR1B antibodies targeting different epitopes .

• Tissue panel evaluation: Test the antibody on a panel of tissues with known differential expression of MTNR1B, such as retina (high expression) versus tissues with lower expected expression .

Implementing these validation strategies will significantly enhance confidence in experimental findings using this antibody.

How can MTNR1B Antibody, FITC conjugated contribute to diabetes research, particularly in relation to the rs10830963 polymorphism?

The MTNR1B antibody, FITC conjugated can significantly advance diabetes research, especially in studying the mechanisms connecting the rs10830963 C/G polymorphism to type 2 diabetes mellitus (T2DM). This polymorphism has been associated with increased T2DM risk through several mechanisms :

• Expression level analysis: The antibody can be used to quantify MTNR1B protein levels in pancreatic islet cells from donors with different genotypes (CC, CG, GG), testing the hypothesis that the G allele increases MTNR1B expression .

• Functional impact studies: Researchers can employ the antibody to track changes in receptor localization and abundance following glucose challenges in cellular models representing different genotypes.

• Pathway visualization: Using multi-color immunofluorescence, the FITC-conjugated antibody can help visualize interactions between MTNR1B and components of insulin secretion pathways, including adenylate cyclase and downstream elements of the cyclic guanine nucleotide pathway .

• Therapeutic targeting assessment: The antibody can be used to evaluate the binding efficiency and specificity of potential MTNR1B antagonists being developed as therapeutic approaches for individuals carrying the G risk allele .

This research approach could provide crucial mechanistic insights into how the rs10830963 polymorphism increases T2DM risk by affecting fasting glucose levels and β-cell function, potentially leading to new therapeutic strategies.

What are the methodological considerations for investigating MTNR1B in circadian rhythm disorders using the FITC-conjugated antibody?

When investigating MTNR1B's role in circadian rhythm disorders using the FITC-conjugated antibody, researchers should consider several methodological aspects:

• Temporal sampling design: MTNR1B expression follows circadian patterns, necessitating systematic sampling across multiple time points in a 24-hour cycle. The FITC-conjugated antibody can be used to map receptor expression fluctuations in relevant tissues.

• Tissue-specific protocols: Different tissues require optimized protocols:

  • For brain tissues (particularly suprachiasmatic nucleus): Use thin sections (5-8μm) and optimized antigen retrieval methods similar to those validated for brain immunohistochemistry .

  • For retinal tissues: Employ specialized fixation protocols to preserve both structure and antigenicity, similar to methods used in validated retinal staining .

• Co-localization studies: Combine MTNR1B antibody with markers for other circadian clock components to establish functional relationships within the circadian timing system.

• Comparative analysis in disease models: Apply the antibody in parallel to samples from normal subjects and those with circadian disorders to identify pathological alterations in receptor expression or localization.

• Quantitative approach: Develop standardized image analysis protocols to quantify MTNR1B immunofluorescence intensity, allowing for objective comparisons between experimental conditions and time points.

These methodological considerations enable rigorous investigation of MTNR1B's contributions to circadian rhythm regulation and its dysregulation in sleep and metabolic disorders.

What quantitative methods are recommended for analyzing MTNR1B expression patterns detected with FITC-conjugated antibodies?

For quantitative analysis of MTNR1B expression detected with FITC-conjugated antibodies, researchers should consider these methodological approaches:

• Fluorescence intensity quantification:

  • Measure mean fluorescence intensity within defined cellular compartments

  • Generate intensity histograms to assess distribution patterns

  • Apply background subtraction using negative control samples

• Co-localization analysis:

  • Calculate Pearson's or Mander's coefficients when examining co-distribution with other proteins

  • Generate intensity correlation plots to assess spatial relationships

• Expression pattern characterization:

  • Categorize expression patterns (membrane, cytoplasmic, nuclear, or mixed)

  • Quantify percentage of cells showing each pattern

  • Measure the relative distribution across cellular compartments

• Comparative statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Consider paired analyses for before/after treatments

  • Use ANOVA for multi-group comparisons with post-hoc tests

• Standardization approaches:

  • Use internal reference standards in each experiment

  • Normalize to housekeeping proteins when appropriate

  • Account for potential variability in antibody lots through standardization controls

These quantitative approaches provide robust frameworks for objective analysis of MTNR1B expression patterns, enabling meaningful comparisons across experimental conditions and between research groups.

How should researchers interpret variations in MTNR1B localization patterns observed with FITC-conjugated antibodies?

When interpreting variations in MTNR1B localization patterns observed using FITC-conjugated antibodies, researchers should consider multiple factors that may influence receptor distribution:

• Physiological state interpretation:

  • Membrane localization typically indicates receptors available for ligand binding

  • Cytoplasmic accumulation may reflect receptor internalization following activation

  • Changes in distribution patterns may correlate with circadian timing or metabolic state

• Technical versus biological variations:

  • Distinguish between technical artifacts and true biological variations through systematic controls

  • Confirm patterns across multiple samples and experimental replicates

  • Validate observations using complementary techniques (e.g., subcellular fractionation)

• Context-dependent localization:

  • MTNR1B normally localizes to the cytoplasm but may show differential distribution in various tissues

  • Altered localization may occur in pathological states, particularly in metabolic disorders

  • Consider how experimental manipulations may affect receptor trafficking

• Correlation with functional outcomes:

  • Relate observed localization patterns to functional measures (e.g., adenylate cyclase activity, cAMP levels)

  • Consider how G-protein coupling status may influence receptor distribution

  • Integrate findings with known MTNR1B signaling mechanisms involving inhibition of adenylate cyclase