NPR3 Antibody, FITC conjugated

What is NPR3 and why is it a significant research target?

NPR3, or Natriuretic Peptide Receptor C, functions as an atrial natriuretic peptide receptor involved in cardiovascular homeostasis. The significance of this receptor lies in its role within the natriuretic peptide family, which elicits crucial vascular, renal, and endocrine effects that maintain blood pressure and extracellular fluid volume regulation . Understanding NPR3 function contributes to cardiovascular disease research, as altered expression or function may correlate with pathological conditions. Researchers target NPR3 to elucidate signaling mechanisms, receptor-ligand interactions, and downstream physiological effects that could inform therapeutic development for conditions such as hypertension, heart failure, and other cardiovascular disorders.

What are the structural characteristics and isoforms of the NPR3 protein?

The NPR3 protein exists in multiple structural variants with distinct molecular properties. According to available data, NPR3 has three isoforms with molecular weights ranging from 37 to 60 kDa, produced through alternative splicing mechanisms . Additionally, post-translational modifications, specifically phosphorylation, can increase the molecular weight to approximately 70 kDa . The human NPR3 consists of 541 amino acids with a calculated molecular weight of 60 kDa . These variations in structure and post-translational modifications potentially influence receptor function, ligand binding affinity, and signaling capabilities, making characterization of specific isoforms relevant for targeted research applications.

What are the key specifications of commercially available NPR3 Antibody, FITC conjugated products?

The commercially available NPR3 Antibody, FITC conjugated products exhibit specific technical characteristics that researchers should consider when selecting reagents. These polyclonal antibodies are typically rabbit-derived IgG isotype, purified using Protein G methods with >95% purity . The immunogen used for production varies by target species: human-reactive antibodies utilize recombinant Human Atrial natriuretic peptide receptor 3 protein (amino acids 36-127) , while rat-reactive antibodies use recombinant Rattus norvegicus Atrial natriuretic peptide receptor 3 protein (amino acids 41-477) . Standard formulation includes preservation with 0.03% Proclin 300 in 50% Glycerol, 0.01M PBS at pH 7.4 . Storage recommendations specify -20°C or -80°C conditions, with cautions against repeated freeze-thaw cycles to maintain antibody performance .

How should researchers validate the specificity of NPR3 Antibody, FITC conjugated before experimental application?

Validating antibody specificity requires a systematic approach to ensure reliable experimental outcomes. Begin with western blot analysis using positive control tissues known to express NPR3 (such as kidney or vascular tissue) alongside negative control samples, verifying bands at expected molecular weights (37-60 kDa and possibly 70 kDa with phosphorylation) . Implement competitive blocking experiments by pre-incubating the antibody with excess recombinant NPR3 protein before staining, which should abolish specific signals. Compare staining patterns with alternative NPR3 antibody clones to confirm consistent localization patterns. For FITC-conjugated antibodies specifically, conduct parallel experiments using unconjugated primary antibody with secondary detection to confirm that conjugation hasn't altered binding properties. Additionally, knockout/knockdown validation provides the strongest specificity evidence—compare staining in wild-type versus NPR3 knockout/knockdown samples to confirm signal absence in depleted specimens.

What is the optimal protocol for using NPR3 Antibody, FITC conjugated in flow cytometry?

The optimal protocol for flow cytometry applications with NPR3 Antibody, FITC conjugated involves several critical methodological steps. Begin with single-cell suspension preparation (approximately 1×10^6 cells per sample) through enzymatic dissociation using collagenase/dispase for tissue samples or gentle trypsinization for adherent cells. Wash cells twice in cold PBS containing 1% BSA, then fix with 2% paraformaldehyde for 15 minutes at room temperature if intracellular staining is required. For membrane-bound NPR3 detection, permeabilization can be omitted. Block non-specific binding using 5% normal serum from the same species as the secondary antibody for 30 minutes. Incubate with NPR3 Antibody, FITC conjugated at experimentally determined optimal concentration (starting dilution 1:100, then titrate as needed) . After 45-60 minutes incubation at 4°C in darkness, wash three times with PBS/BSA buffer. Include appropriate compensation controls for multicolor panels and isotype-matched FITC-conjugated control antibodies to establish background fluorescence thresholds. Analyze using 488nm excitation laser and appropriate emission filters (typically 530/30nm) for FITC detection.

How can NPR3 Antibody, FITC conjugated be utilized in immunofluorescence microscopy?

Implementing NPR3 Antibody, FITC conjugated in immunofluorescence microscopy requires specific methodological considerations to obtain optimal visualization. Begin with appropriate sample preparation: fix tissue sections or cultured cells with 4% paraformaldehyde for 15-20 minutes, followed by permeabilization with 0.1-0.3% Triton X-100 if intracellular epitopes are targeted. Block non-specific binding with 5-10% normal serum and 1% BSA for 1 hour at room temperature. Apply the FITC-conjugated NPR3 antibody at experimentally optimized dilutions, typically starting at 1:100-1:400 , and incubate overnight at 4°C in a humidified, dark chamber. For tissue sections, antigen retrieval may be necessary, with recommended protocols using TE buffer at pH 9.0 or citrate buffer at pH 6.0 . Include DAPI (1:1000) during the final wash steps for nuclear counterstaining. Mount using anti-fade mounting medium specific for fluorescence preservation. Capture images using confocal or epifluorescence microscopy with appropriate excitation (488nm) and emission (515-530nm) filters for FITC visualization. Include controls for autofluorescence (unstained sample) and non-specific binding (isotype control) in parallel experiments.

What are common sources of background signal when using NPR3 Antibody, FITC conjugated, and how can they be mitigated?

Background signal challenges with FITC-conjugated antibodies can originate from multiple sources that require specific mitigation strategies. Autofluorescence from tissues (particularly collagen-rich cardiovascular specimens) can be reduced by including a brief incubation (10 minutes) with 0.1% Sudan Black B in 70% ethanol after antibody staining, or using commercially available autofluorescence quenching reagents. Non-specific antibody binding can be addressed by optimizing blocking conditions—try increasing blocking serum concentration to 10% and extending blocking time to 2 hours, or adding 0.1-0.3% Triton X-100 to blocking solutions. FITC's inherent sensitivity to photobleaching necessitates minimal light exposure during all procedural steps, sample storage in darkness, and rapid image acquisition. For flow cytometry applications, dead cell contribution to background can be minimized using viability dyes and implementing stringent gating strategies. If tissue-specific background persists, consider counterstaining with alternative fluorophores to spectrally separate signals from autofluorescence. Additionally, titrate antibody concentrations systematically (usually between 1:100-1:400) to determine optimal signal-to-noise ratio for each experimental system.

How should researchers address potential cross-reactivity with other natriuretic peptide receptors?

Addressing potential cross-reactivity with related natriuretic peptide receptors requires implementation of multiple validation controls and analytical approaches. First, conduct comprehensive in silico analysis comparing immunogen sequence (amino acids 36-127 for human NPR3 or 41-477 for rat NPR3) against NPR1 and NPR2 sequences to identify regions of homology that might predispose to cross-reactivity. Experimentally, perform parallel staining in models with differential expression of NPR family members, such as cell lines with known expression profiles of specific receptors. Implement competition assays using recombinant NPR1, NPR2, and NPR3 proteins to assess binding specificity. For definitive validation, utilize siRNA knockdown or CRISPR knockout models specifically targeting NPR3 while monitoring other family members to confirm that signal reduction correlates exclusively with NPR3 depletion. Western blot analysis can provide additional confirmation through molecular weight discrimination, as NPR1 (~130 kDa), NPR2 (~120 kDa), and NPR3 (37-60 kDa) exhibit distinct sizes. If cross-reactivity is detected, consider alternative antibody clones or epitope-specific antibodies targeting unique regions of NPR3 with minimal sequence homology to other family members.

What are the critical storage and handling considerations to maintain NPR3 Antibody, FITC conjugated performance?

Maintaining optimal performance of FITC-conjugated NPR3 antibodies requires adherence to specific storage and handling protocols that preserve both antibody integrity and fluorophore activity. Store antibody at recommended temperatures (-20°C or -80°C) in manufacturer-supplied buffer containing stabilizers (50% glycerol, 0.01M PBS, pH 7.4) . Minimize freeze-thaw cycles by preparing small working aliquots upon receipt—FITC conjugates typically tolerate no more than 3-5 freeze-thaw cycles before significant performance degradation occurs. Protect from light at all stages of handling by wrapping storage containers in aluminum foil and working under reduced ambient lighting conditions, as FITC is particularly susceptible to photobleaching. When preparing dilutions, use high-quality, sterile-filtered buffers free of contaminating proteins that might compete for binding. For long-term storage beyond manufacturer recommendations (typically one year) , monitor performance through regular quality control testing against known positive samples. The addition of protein stabilizers (0.1% BSA) to working dilutions can enhance antibody stability during experimental procedures. Always maintain sample chain of cold, handling antibodies on ice when out of storage, and return to appropriate temperature conditions promptly after use.

How can NPR3 Antibody, FITC conjugated be employed in multiplex immunophenotyping of cardiovascular tissues?

Implementing NPR3 Antibody, FITC conjugated in multiplex immunophenotyping requires strategic panel design and methodological optimization for comprehensive cardiovascular tissue characterization. Begin with spectral compatibility planning—FITC excitation/emission properties (excitation maximum ~495nm, emission maximum ~520nm) allow complementary pairing with fluorophores like PE (578nm), APC (660nm), and Pacific Blue (455nm) for minimal spillover in multiplex panels. For tissue-based multiplexing, implement sequential staining protocols with careful antibody order consideration; typically apply FITC-conjugated antibodies early in the sequence as they exhibit greater susceptibility to photobleaching. When analyzing endothelial-vascular interactions, combine NPR3-FITC with endothelial markers (CD31-PE), smooth muscle indicators (α-SMA-APC), and macrophage markers (CD68-Pacific Blue) to assess cardiovascular tissue microenvironment comprehensively. For quantitative co-localization analysis, utilize specialized image analysis software with colocalization algorithms such as Manders' or Pearson's correlation coefficients. Employ multispectral imaging systems with spectral unmixing capabilities to resolve overlapping fluorophore emissions in densely labeled specimens. Consider implementing automated machine learning-based quantification approaches for unbiased assessment of marker co-expression patterns across tissue regions with variable NPR3 expression levels.

What methodological approaches enable quantitative assessment of NPR3 receptor density using FITC-conjugated antibodies?

Quantitative assessment of NPR3 receptor density utilizing FITC-conjugated antibodies requires standardized approaches that enable reliable numerical determination of receptor abundance. For flow cytometry-based quantification, implement Quantibrite™ or similar fluorescence calibration beads with known FITC molecules per bead to establish a standard curve correlating mean fluorescence intensity (MFI) to absolute fluorophore numbers. Apply the antibody at saturating concentrations (determined through titration experiments) to ensure all available epitopes are bound. Determine the fluorophore:protein (F:P) ratio of the specific antibody lot from manufacturer specifications to calculate antibody binding sites from FITC fluorescence values. For microscopy-based quantification, utilize digital image analysis with calibrated fluorescence intensity standards in each experimental session. Apply thresholding algorithms consistently across experimental groups, measuring integrated density values normalized to cell number or tissue area. For super-resolution microscopy applications, implement single-molecule localization techniques such as STORM or PALM, where individual NPR3 molecules can be counted directly after appropriate controls for antibody specificity and binding stoichiometry. Consider using quantitative PCR and western blotting in parallel experiments to correlate protein expression levels with fluorescence intensity measurements for comprehensive receptor density validation.

How can researchers investigate NPR3 dynamics in live cell imaging using FITC-conjugated antibodies?

Investigating NPR3 dynamics in live cell imaging with FITC-conjugated antibodies presents technical challenges that require specialized methodological approaches. Because conventional antibodies cannot penetrate intact cell membranes, limit live cell applications to membrane-expressed NPR3 detection by using antibodies targeting extracellular domains (amino acids 36-127 for human NPR3) . Implement Fragment antigen-binding (Fab) preparation through enzymatic digestion of the FITC-conjugated antibody to reduce size and potential receptor crosslinking that might alter normal trafficking. For pulse-chase experiments, expose cells to the FITC-labeled Fab fragments at 4°C (to permit binding without internalization), wash extensively, then warm to 37°C while initiating time-lapse imaging to track receptor internalization kinetics. Employ spinning disk confocal microscopy with sensitive EM-CCD cameras and minimal light exposure (using neutral density filters) to reduce phototoxicity while maintaining adequate signal. Consider photobleaching approaches such as Fluorescence Recovery After Photobleaching (FRAP) to assess receptor lateral mobility within membranes. For extended imaging sessions, supplement culture media with ascorbic acid (100μM) as an antioxidant to reduce phototoxicity. To distinguish between internal and newly synthesized receptors, combine with complementary approaches like SNAP-tag or HaloTag labeling of recombinant NPR3 constructs for orthogonal visualization of receptor populations.

How do researchers interpret variable NPR3 expression patterns across different cardiovascular cell types?

Interpreting variable NPR3 expression patterns across cardiovascular cell types requires integrated analysis of cellular context, physiological state, and methodological considerations. Establish baseline expression through systematic comparison of endothelial cells, vascular smooth muscle cells, cardiomyocytes, and cardiac fibroblasts using consistent staining protocols and acquisition parameters. Quantify expression using standardized metrics (mean fluorescence intensity, integrated density values, or receptor number per cell) with appropriate statistical analysis for inter-population comparisons. When observing differential expression, consider physiological relevance—endothelial cells typically express higher NPR3 levels correlating with their role in mediating natriuretic peptide clearance from circulation, while cardiomyocytes may show context-dependent expression relating to cardiac stress responses. Validate flow cytometry findings through complementary techniques such as quantitative PCR, western blotting, or in situ hybridization to distinguish between transcriptional and post-transcriptional regulation mechanisms. For heterogeneous tissue analysis, implement computational deconvolution algorithms that can resolve cell type-specific contributions to bulk expression measurements. Consider experimental conditions that might influence receptor expression, including culture confluence, passage number for in vitro studies, or disease state and tissue region for in vivo analyses, as these factors significantly impact interpretation of observed expression patterns.

How should researchers integrate NPR3 expression data with functional assays to establish physiological relevance?

Establishing physiological relevance of NPR3 expression patterns requires methodical integration of expression data with functional assessments through complementary experimental approaches. Start by correlating NPR3 expression levels (quantified using the FITC-conjugated antibody) with functional readouts of natriuretic peptide signaling, such as cGMP production, calcium mobilization, or specific downstream kinase activation. Implement pharmacological manipulation using selective NPR3 agonists (cANF4-23) or antagonists (M372049) while monitoring both receptor expression and functional outcomes to establish causative relationships. Design genetic modulation experiments using siRNA knockdown, CRISPR-Cas9 knockout, or overexpression systems with titrated expression levels to create dose-response relationships between receptor abundance and functional parameters. For cardiovascular applications specifically, correlate NPR3 expression with physiological measurements such as vessel tone, cardiomyocyte contractility, or endothelial permeability using ex vivo tissue preparations or in vitro cellular systems. Extend this integration to disease-relevant contexts by comparing NPR3 expression between normal and pathological samples (hypertension, heart failure, atherosclerosis) while simultaneously assessing functional alterations in corresponding models. Implement mathematical modeling approaches that incorporate quantitative expression data as parameters in signaling networks to predict functional outcomes, then validate these predictions experimentally to establish robust structure-function relationships.