NREP Antibody, FITC conjugated

What is NREP and why is it an important research target?

NREP (Neuronal regeneration-related protein), also known as Neuronal protein 3.1 or Protein p311, is a significant protein of interest in neurological and cardiovascular research. The human NREP protein (UniprotID: Q16612) functions as a neuronal regeneration-related protein with implications in cellular development pathways. As identified in product documentation, NREP is associated with aliases including C5orf13 and P311, suggesting its diverse nomenclature in research literature . The protein's involvement in cardiovascular research areas makes FITC-conjugated antibodies against NREP particularly valuable for investigating its expression patterns and localization in various tissue and cell types.

NREP research is distinct from but may share methodological approaches with other neuronal protein studies, such as those involving Neuropilin-2 (NRP2), which is a single-pass multifunctional transmembrane glycoprotein playing roles in cell development, immunity, cancer development, and angiogenesis . Understanding the fundamental properties of NREP provides context for designing experiments using NREP Antibody, FITC conjugated.

What is the significance of FITC conjugation for NREP antibody applications?

FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescent visualization capabilities for NREP antibodies, eliminating the need for secondary antibody detection steps in many experimental protocols. The FITC conjugate emits green fluorescence when excited with appropriate wavelengths, allowing for direct detection of NREP protein in applications such as flow cytometry, immunofluorescence microscopy, and fluorescence-based immunoassays.

Similar to other FITC-conjugated antibodies, such as FITC-labeled anti-CD45 antibodies used in flow cytometry , NREP antibody with FITC conjugation facilitates visualization of target proteins in intact cells and tissues. The significance of this conjugation extends to pH-dependent experimental designs, as FITC fluorescence properties can vary with pH, potentially providing additional experimental readouts in studies involving pH changes . The preservation of antibody specificity while gaining fluorescent detection capabilities makes FITC-conjugated NREP antibodies valuable tools for multiple research applications.

How should NREP Antibody, FITC conjugated be stored and handled to maintain optimal activity?

Proper storage and handling of NREP Antibody, FITC conjugated is critical to maintaining its immunoreactivity and fluorescence properties. The antibody should be stored at -20°C or -80°C upon receipt, with repeated freeze-thaw cycles strictly avoided to prevent degradation of both the antibody and the FITC conjugate . The antibody is supplied in a protective buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative, which helps maintain stability during storage .

When working with the antibody:

• Thaw aliquots completely before use, but keep cold during experimental preparation

• Protect from prolonged light exposure to prevent photobleaching of the FITC fluorophore

• Use appropriate controls (isotype controls) when designing experiments

• Maintain recommended working dilutions based on application type

Similar to handling protocols for other FITC-conjugated antibodies, such as those employed with FITC-based pHLIP conjugates in cancer cell treatments , minimizing light exposure during storage and experimental procedures is essential for preserving the fluorescent signal strength.

What are the optimal protocols for using NREP Antibody, FITC conjugated in flow cytometry experiments?

For flow cytometry applications with NREP Antibody, FITC conjugated, researchers should implement the following optimized protocol based on experimental evidence with similar FITC-conjugated antibodies:

• Cell Preparation:

  • Harvest cells (1×10^6 cells per sample) and wash twice with cold PBS containing 2% FBS

  • Resuspend cells in 100 μL flow cytometry buffer (PBS with 2% FBS and 0.1% sodium azide)

• Antibody Staining:

  • Add NREP Antibody, FITC conjugated at the optimized concentration (typically starting at 5-10 μg/mL for titration)

  • Incubate for 30-45 minutes at 4°C in the dark

  • Wash twice with 2 mL flow cytometry buffer

• Controls and Analysis:

  • Include unstained cells, isotype control-FITC, and single-color controls for compensation

  • Acquire data using 488 nm laser excitation and appropriate emission filters (typically 525/40 nm)

  • Analyze data using standard flow cytometry software with appropriate gating strategies

This approach mirrors protocols established for other FITC-conjugated antibodies such as anti-CD45 FITC antibody , while accounting for the specific properties of NREP antibody. For detecting cellular surface expression, investigators should consider non-permeabilizing conditions, while intracellular NREP detection would require appropriate fixation and permeabilization steps.

How can NREP Antibody, FITC conjugated be utilized in dual immunofluorescence staining protocols?

For dual immunofluorescence applications, NREP Antibody, FITC conjugated can be effectively combined with other fluorophore-labeled antibodies, provided appropriate spectral separation considerations are implemented:

Protocol for Dual Immunofluorescence with NREP Antibody, FITC conjugated:

• Sample Preparation:

  • Fix cells/tissue with 4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 if intracellular staining is required

  • Block with 5% normal serum (species matched to secondary antibody) for 1 hour

• Sequential Staining Approach:

  • First incubation: Apply non-FITC primary antibody overnight at 4°C

  • Wash 3× with PBS

  • Second incubation: Apply appropriate secondary antibody (using spectrally distinct fluorophore like Cy3, Alexa 594, or Alexa 647)

  • Wash 3× with PBS

  • Third incubation: Apply NREP Antibody, FITC conjugated (diluted to 2-5 μg/mL) for 1-2 hours at room temperature

  • Wash 3× with PBS

  • Mount with anti-fade medium containing DAPI for nuclear counterstaining

• Controls:

  • Single antibody controls to assess bleed-through

  • Isotype controls for both antibodies

  • Secondary antibody-only controls

This sequential staining approach minimizes potential cross-reactivity issues, particularly if the other primary antibody is from rabbit origin (similar to the NREP antibody host species). Methodologically, this approach is similar to techniques used with other FITC-conjugated antibodies in studies examining immunogenic epitopes .

What are the critical considerations when using NREP Antibody, FITC conjugated in pH-dependent experimental designs?

When utilizing NREP Antibody, FITC conjugated in experiments involving pH variations, researchers must account for several critical factors:

• FITC Fluorescence pH Sensitivity:

  • FITC fluorescence intensity decreases at lower pH values

  • Maximum fluorescence occurs around pH 8-9

  • Significant quenching occurs below pH 6.0

• Experimental Calibration:

  • Include pH calibration standards in each experiment

  • Use pH-insensitive dyes (e.g., Alexa Fluor 488) as controls in parallel samples

• Data Analysis Considerations:

  • Apply correction factors based on pH calibration curves

  • Normalize FITC signals to pH-insensitive reference markers

  • Consider ratiometric approaches for accurate quantification

Research with FITC-pHLIP conjugates demonstrates how pH-dependent changes can affect antibody recruitment and cellular fluorescence levels . Treatment of MDA-MB-231 cells with 1 μM FITC-based pHLIP conjugates at different pH conditions (pH 7.4 versus pH 6.0) resulted in pH-dependent differences in antibody recruitment and fluorescence intensity . Similar considerations would apply to NREP Antibody, FITC conjugated when used in experimental designs involving pH variations.

How can specificity of NREP Antibody, FITC conjugated be validated in experimental systems?

Validating the specificity of NREP Antibody, FITC conjugated requires a multi-faceted approach incorporating several complementary techniques:

• Positive and Negative Cell Type Controls:

  • Test antibody on cells known to express NREP at different levels

  • Include negative control cells with minimal or no NREP expression

  • Compare staining patterns with mRNA expression data from databases

• Blocking Experiments:

  • Pre-incubate antibody with recombinant NREP protein (immunogen)

  • Compare staining intensity between blocked and unblocked antibody

  • Substantial reduction in signal indicates specific binding

• siRNA/shRNA Knockdown Validation:

  • Transfect cells with NREP-targeting siRNA/shRNA

  • Confirm knockdown by RT-qPCR

  • Demonstrate reduced antibody staining in knockdown cells

• Cross-Reactivity Assessment:

  • Test antibody against closely related proteins (if available)

  • Evaluate potential cross-reactivity with structural homologs

These approaches reflect standard validation techniques used for antibodies, including FITC-conjugated ones. For instance, specificity validation approaches similar to those demonstrated with anti-FITC antibodies, where researchers confirmed that anti-FITC-A647 specifically bound to FITC-based conjugates and not to the cells themselves , should be employed for NREP Antibody, FITC conjugated.

What are the optimal fixation and permeabilization protocols when using NREP Antibody, FITC conjugated for immunocytochemistry?

For immunocytochemistry applications with NREP Antibody, FITC conjugated, optimization of fixation and permeabilization protocols is crucial for balancing epitope preservation, cellular morphology, and antibody accessibility:

Recommended Fixation Methods (in order of preference):

• 4% Paraformaldehyde (PFA):

  • Optimal for preserving FITC fluorescence

  • Fix cells for 15 minutes at room temperature

  • Wash 3× with PBS

• Methanol/Acetone (1:1):

  • Alternative for certain applications requiring different epitope exposure

  • Fix cells for 10 minutes at -20°C

  • Air dry briefly before rehydrating with PBS

Permeabilization Options:

• Triton X-100:

  • Use 0.1-0.3% in PBS for 5-10 minutes

  • Provides good permeabilization but may affect membrane proteins

• Saponin:

  • Use 0.1-0.5% in PBS

  • Milder detergent, better for preserving membrane structures

  • Must be present throughout all subsequent steps

• Digitonin:

  • Use 10-50 μg/mL for selective plasma membrane permeabilization

  • Useful for distinguishing cytoplasmic versus nuclear localization

Researchers should carefully evaluate which combination works best for their specific experimental system, as fixation can affect FITC fluorescence and NREP epitope accessibility. Similar considerations have been documented in studies utilizing other FITC-conjugated antibodies, where fixation conditions significantly impacted fluorescence intensity and staining patterns .

How can researchers troubleshoot weak or non-specific signals when using NREP Antibody, FITC conjugated?

When encountering weak or non-specific signals with NREP Antibody, FITC conjugated, researchers should implement a systematic troubleshooting approach:

For Weak Signal:

• Antibody Concentration Optimization:

  • Titrate antibody in 2-fold serial dilutions

  • Test range from 1-20 μg/mL to determine optimal signal-to-noise ratio

• Incubation Conditions:

  • Extend incubation time (up to overnight at 4°C)

  • Optimize temperature (4°C, room temperature, or 37°C)

• Signal Amplification Options:

  • Consider anti-FITC secondary antibodies labeled with brighter fluorophores

  • Implement tyramide signal amplification compatible with FITC wavelengths

• Sample Preparation Improvements:

  • Test different fixation protocols to better preserve epitopes

  • Optimize antigen retrieval methods if applicable

For Non-specific Signal:

• Blocking Optimization:

  • Increase blocking agent concentration (5-10% normal serum)

  • Add 0.1-0.3% Triton X-100 to blocking buffer

  • Consider specialized blocking agents (e.g., Image-iT FX Signal Enhancer)

• Washing Stringency:

  • Increase number of washes (5-6 times)

  • Extend washing time (10-15 minutes per wash)

  • Add 0.05-0.1% Tween-20 to wash buffer

• Antibody Validation:

  • Perform absorption controls with recombinant NREP

  • Test on known negative cell types or tissues

• Autofluorescence Reduction:

  • Treat samples with 0.1-1% sodium borohydride

  • Use Sudan Black B (0.1-0.3%) to quench lipofuscin autofluorescence

This troubleshooting approach is derived from established methodologies for optimizing fluorescently labeled antibodies, including techniques utilized in studies with other FITC-conjugated antibodies .

How can NREP Antibody, FITC conjugated be used in quantitative studies of NREP expression across different cell types?

For quantitative assessment of NREP expression using FITC-conjugated antibodies, researchers should implement a standardized approach incorporating calibration standards and appropriate controls:

Quantitative Flow Cytometry Protocol:

• Calibration Setup:

  • Use FITC Calibration Beads with defined molecules of equivalent soluble fluorochrome (MESF)

  • Generate standard curve relating fluorescence intensity to MESF values

  • Include Rainbow Calibration Particles for instrument standardization

• Sample Processing:

  • Prepare single-cell suspensions from different cell types/tissues

  • Stain with NREP Antibody, FITC conjugated at optimized concentration

  • Include unstained and isotype controls for each cell type

• Data Acquisition and Analysis:

  • Collect minimum of 10,000 events per sample

  • Gate populations based on forward/side scatter properties

  • Determine median fluorescence intensity (MFI) for each sample

  • Convert MFI to MESF using calibration curve

  • Calculate antibody binding capacity using MESF values

• Normalization Strategies:

  • Normalize to cell surface area for comparing different cell types

  • Use internal reference proteins for relative quantification

  • Apply appropriate statistical analysis for multiple comparisons

This approach enables robust comparison of NREP expression levels across different cell types or experimental conditions. Similar quantitative methodologies have been demonstrated with other FITC-conjugated antibodies, such as anti-CD45 antibodies used in clinical flow cytometry applications .

What are the considerations for using NREP Antibody, FITC conjugated in live cell imaging experiments?

Live cell imaging with NREP Antibody, FITC conjugated presents unique challenges and requires specific optimization strategies:

Protocol Considerations:

• Antibody Delivery Options:

  • Direct addition to culture medium (for surface epitopes)

  • Microinjection (for intracellular targets)

  • Cell-penetrating peptide conjugation (for intracellular delivery)

• Imaging Parameters:

  • Use minimal laser power/exposure time to reduce phototoxicity

  • Implement time-lapse intervals appropriate for biological process

  • Consider confocal or spinning disk microscopy for optimal resolution

• Physiological Considerations:

  • Maintain cells at 37°C with appropriate CO₂ levels

  • Use phenol red-free media to reduce background

  • Supplement media with oxygen scavengers to reduce phototoxicity

• Controls and Validation:

  • Include membrane-impermeable viability dyes

  • Monitor cellular morphology and behavior

  • Compare with fixed-cell staining patterns

Research with FITC-based pHLIP conjugates demonstrates how fluorescently labeled molecules can be used in live cell experiments to detect antibody recruitment . When working with pH-sensitive applications, researchers should be aware that FITC fluorescence properties can vary with environmental pH, requiring appropriate controls and calibration .

How can NREP Antibody, FITC conjugated be incorporated into tissue microarray (TMA) analysis for high-throughput research?

Tissue Microarray (TMA) analysis with NREP Antibody, FITC conjugated enables high-throughput examination of NREP expression across multiple tissue samples simultaneously. The following protocol optimizes this application:

TMA Protocol with NREP Antibody, FITC conjugated:

• TMA Preparation:

  • Deparaffinize and rehydrate sections according to standard protocols

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Block endogenous peroxidase activity if using brightfield conversion

• Antibody Application:

  • Apply optimized concentration of NREP Antibody, FITC conjugated

  • Incubate in humidity chamber for 1-2 hours at room temperature or overnight at 4°C

  • Wash thoroughly with PBS-T (PBS + 0.1% Tween-20)

• Signal Detection Options:

  • Direct fluorescence: Mount with anti-fade medium containing DAPI

  • Signal amplification: Apply anti-FITC antibody conjugated to HRP, followed by tyramide signal amplification

  • Brightfield conversion: Use anti-FITC-HRP followed by DAB chromogen development

• Image Acquisition and Analysis:

  • Use automated slide scanner with appropriate filters

  • Implement tissue segmentation algorithms

  • Quantify NREP expression using H-score, Allred score, or continuous intensity measurements

• Data Integration:

  • Correlate NREP expression with clinical parameters

  • Perform statistical analysis across tissue types or disease states

  • Integrate with other molecular markers for pathway analysis

This approach enables systematic evaluation of NREP expression across large sample cohorts while maintaining standardized staining conditions. Similar high-throughput approaches have been utilized with other fluorescently-labeled antibodies in biomarker research contexts .

How does NREP Antibody, FITC conjugated compare with other detection methods for NREP protein?

When evaluating NREP detection methods, researchers should consider the following comparative analysis of NREP Antibody, FITC conjugated versus alternative approaches:

What are the known limitations of using NREP Antibody, FITC conjugated in research applications?

Researchers should be aware of several limitations when working with NREP Antibody, FITC conjugated:

• Photobleaching Susceptibility:

  • FITC fluorophore is more prone to photobleaching than newer dyes

  • Limit exposure to light during storage and experimental procedures

  • Consider alternative conjugates (Alexa Fluor 488) for prolonged imaging

• pH Sensitivity:

  • FITC fluorescence decreases significantly at acidic pH

  • May complicate interpretation in acidic cellular compartments

  • Requires careful control design in experiments involving pH changes

• Spectral Limitations:

  • Relatively broad emission spectrum limits multiplexing capabilities

  • Potential bleed-through with other green-yellow fluorophores

  • Autofluorescence in green channel can interfere with detection

• Fixation Compatibility:

  • Certain fixatives may diminish FITC signal

  • Glutaraldehyde should be avoided or used at minimal concentrations

  • Optimize fixation protocols for specific applications

• Potential Effects on Antibody Function:

  • FITC conjugation might affect binding kinetics or affinity

  • Higher concentrations may be needed compared to unconjugated antibody

  • Batch-to-batch variation in conjugation efficiency

These limitations mirror challenges encountered with other FITC-conjugated antibodies, such as those documented in studies using FITC-based pHLIP conjugates in cancer cell treatments , where pH sensitivity had to be carefully accounted for in experimental design.

How should researchers interpret discrepancies between NREP Antibody, FITC conjugated results and other NREP detection methods?

When encountering discrepancies between results obtained with NREP Antibody, FITC conjugated and other detection methods, researchers should implement a systematic analytical approach:

• Technical Validation:

  • Repeat experiments with appropriate positive and negative controls

  • Verify antibody lot performance with standard samples

  • Test multiple detection methods on identical samples

• Biological Explanations:

  • Post-translational modifications: Different antibodies may recognize different epitopes affected by PTMs

  • Protein isoforms: Check if discrepancies correlate with known NREP isoforms

  • Protein-protein interactions: Consider if binding partners mask epitopes

  • Subcellular localization: Compare detection methods' ability to access different cellular compartments

• Methodological Considerations:

  • Sensitivity thresholds: Determine detection limits for each method

  • Linear dynamic range: Assess if quantification falls within linear range

  • Sample preparation effects: Evaluate how different preparations affect epitope availability

• Resolution Strategy:

  • Use orthogonal approaches to validate key findings

  • Report discrepancies transparently in publications

  • Consider biological significance of differences between detection methods

This analytical framework helps distinguish genuine biological variations from technical artifacts. Similar approaches have been applied when resolving discrepancies between results obtained with different fluorescently labeled antibodies targeting the same protein .

How is NREP Antibody, FITC conjugated contributing to advancing understanding of NREP biology?

NREP Antibody, FITC conjugated provides researchers with a powerful tool for directly visualizing NREP protein expression and localization in diverse experimental contexts. The direct fluorescent conjugation enables real-time observation in live cells, facilitating studies of dynamic processes involving NREP that were previously challenging to investigate. By enabling more efficient detection protocols without secondary antibodies, this reagent has streamlined research workflows while maintaining the specificity needed for reliable NREP detection.

The applications of NREP Antibody, FITC conjugated span from basic research investigating NREP's role in neuronal regeneration to more applied studies in cardiovascular research areas . Similar to how FITC-conjugated antibodies have advanced other fields, such as cancer immunotherapy research through detection of cell surface epitopes , NREP Antibody, FITC conjugated is helping to illuminate the multifaceted functions of NREP in normal physiology and disease states through both static and dynamic visualization approaches.

What emerging technologies might enhance the utility of NREP Antibody, FITC conjugated in future research?

Several emerging technologies show promise for expanding the utility of NREP Antibody, FITC conjugated in future research applications:

• Super-Resolution Microscopy:

  • STORM/PALM techniques can overcome diffraction limits

  • Potential for nanoscale localization of NREP within cellular structures

  • May reveal previously undetectable NREP distribution patterns

• Spatial Transcriptomics Integration:

  • Combining NREP protein detection with spatial transcriptomics

  • Correlating protein expression with transcriptional programs

  • Multi-omic spatial profiling at single-cell resolution

• Microfluidic Applications:

  • High-throughput screening of NREP expression in response to stimuli

  • Single-cell analysis of NREP dynamics

  • Droplet-based assays for quantitative protein measurements

• Advanced Live Cell Technologies:

  • Photoswitchable FITC derivatives for pulse-chase experiments

  • Integration with optogenetic tools for simultaneous manipulation and observation

  • Correlative light-electron microscopy for ultrastructural context

• AI-Enhanced Image Analysis:

  • Deep learning algorithms for automated NREP detection and quantification

  • Pattern recognition for identifying novel NREP distribution signatures

  • Predictive modeling of NREP dynamics based on image time series

These technological advances parallel developments seen with other fluorescently labeled antibodies, where integration with cutting-edge methodologies has continuously expanded their research applications .

What are the most promising research areas for application of NREP Antibody, FITC conjugated in interdisciplinary studies?

NREP Antibody, FITC conjugated offers significant potential for interdisciplinary research spanning several high-impact areas:

• Neurodegenerative Disease Research:

  • Investigating NREP's role in neuronal regeneration mechanisms

  • Correlation of NREP expression with disease progression

  • Screening potential therapeutics targeting NREP pathways

• Cardiovascular Medicine:

  • Examining NREP in cardiac tissue remodeling

  • Vascular development and angiogenesis studies

  • Biomarker development for cardiovascular conditions

• Regenerative Medicine:

  • NREP's potential role in tissue regeneration

  • Stem cell differentiation and programming

  • Biomaterial development incorporating NREP-related signaling

• Cancer Biology:

  • NREP expression in tumor microenvironments

  • Correlation with invasiveness and metastatic potential

  • Potential therapeutic targeting similar to approaches used with other membrane proteins

• Developmental Biology:

  • NREP dynamics during embryonic development

  • Tissue patterning and organogenesis

  • Evolutionary conservation of NREP functions