NPAS2 Antibody, FITC conjugated

What is NPAS2 and why is it important in neurological and immunological research?

NPAS2 (Neuronal PAS Domain Protein 2) is a core component of the molecular clock that functions as a transcription factor. It is highly expressed in reward- and stress-related brain regions, particularly the striatum . NPAS2 plays a critical role in regulating circadian rhythms and has been implicated in mood-related behaviors, including anxiety .

Recent research has expanded our understanding of NPAS2's functions beyond the central nervous system. It has been shown to inhibit macrophage activation and regulate m6A methylation processes involved in diabetic nephropathy (DN) . Additionally, NPAS2 has been identified as a potential modulator of chemotherapy resistance in lung adenocarcinoma through its role in DNA damage repair mechanisms .

The multifaceted functions of NPAS2 make antibodies against this protein valuable tools for investigating circadian rhythm disorders, psychiatric conditions, inflammatory responses, and cancer mechanisms.

What experimental applications are most suitable for NPAS2 Antibody, FITC conjugated?

NPAS2 Antibody, FITC conjugated is optimized for the following applications:

• Flow Cytometry: The FITC conjugation allows for direct detection of NPAS2 expression in cells, making it ideal for quantitative analysis of NPAS2 levels in heterogeneous cell populations, particularly in macrophages where NPAS2 has been shown to mediate inflammatory responses .

• Immunofluorescence Microscopy: This antibody enables visualization of NPAS2 localization in tissue sections or cultured cells. This is particularly useful for studying NPAS2 expression in specific brain regions like the striatum, where it influences mood-related behaviors .

• ChIP-Based Applications: Modified protocols can adapt this antibody for chromatin immunoprecipitation studies to identify NPAS2 target genes, such as GABAA receptor subunit genes (Gabra) .

• Confocal Microscopy: The FITC conjugation provides excellent signal-to-noise ratio for high-resolution imaging of NPAS2 localization in subcellular compartments.

For optimal results, sample preparation should account for the circadian expression pattern of NPAS2, with consideration of time-point collection (such as ZT4 and ZT16 as used in published protocols) .

How can NPAS2 Antibody, FITC conjugated be used to investigate the role of NPAS2 in diabetic nephropathy and macrophage activation?

Recent findings have revealed that NPAS2 plays a significant role in diabetic nephropathy through regulation of macrophage activation and glycolysis. A FITC-conjugated NPAS2 antibody can be employed in the following research approaches:

• Flow Cytometric Analysis of Macrophage Populations:

  • Identify and quantify NPAS2 expression in different macrophage subpopulations (M1 vs. M2) isolated from kidney tissue or bone marrow-derived macrophages (BMDMs)

  • Correlate NPAS2 expression levels with macrophage activation status in diabetic nephropathy models

• Multiplex Immunofluorescence Staining:

  • Co-localize NPAS2 with markers of glycolysis (HK1, PFKFB3) or inflammation (IL-1β, TNF-α)

  • Visualize the relationship between NPAS2 expression and Hif-1α signaling in kidney tissue sections

• Experimental Protocol for Macrophage NPAS2 Analysis:

Research has shown that Fto overexpression reduces m6A modification of Npas2 mRNA in macrophages through a Prrc2a-dependent mechanism, decreasing its stability. This process mediates inflammation and glycolysis in M1 macrophage activation by regulating the Hif-1α signaling pathway . The FITC-conjugated antibody enables direct visualization of these relationships in experimental models.

What approaches can be used to study NPAS2's role in chemotherapy resistance using FITC-conjugated antibodies?

NPAS2 has been identified as a potential mediator of chemoresistance in lung adenocarcinoma (LUAD) through its interaction with DNA damage repair pathways . NPAS2 Antibody, FITC conjugated can be utilized to investigate this mechanism through the following approaches:

• Monitoring NPAS2 Expression in Response to Chemotherapy:

  • Track changes in NPAS2 expression levels in LUAD cells before and after cisplatin treatment using flow cytometry

  • Correlate NPAS2 levels with γH2AX accumulation (a marker of DNA damage) in single-cell analysis

• High-Content Imaging for DNA Damage Response:

  • Perform time-course analysis of NPAS2 localization and expression following chemotherapy in fixed cells

  • Co-stain with markers of homology-directed repair to assess NPAS2's role in DNA repair mechanisms

• Experimental Protocol for Chemoresistance Studies:

Research has demonstrated that NPAS2 binds to and enhances the stability of H2AX mRNA, thereby decreasing the sensitivity of tumor cells to chemotherapy by augmenting DNA damage repair . Using the FITC-conjugated NPAS2 antibody enables direct visualization of this relationship in experimental models of chemoresistance.

What are the optimal protocols for NPAS2 immunofluorescence detection in brain tissue sections?

When using NPAS2 Antibody, FITC conjugated for neurological research, special considerations must be made due to NPAS2's circadian expression patterns and region-specific distribution:

• Tissue Collection and Fixation Protocol:

• Staining Protocol Optimization:

  • Heat-mediated antigen retrieval (10mM sodium citrate, pH 6.0) significantly improves NPAS2 detection in fixed tissue

  • Extended primary antibody incubation (48h at 4°C) may be necessary for penetration into thicker sections

  • For ventral striatum/NAc studies, use tyramide signal amplification to enhance detection of low NPAS2 expression levels

• Controls and Validation:

  • Include Npas2 null mutant mice tissues as negative controls

  • Validate staining pattern using tissues with known high expression (striatum) and low expression (cerebellum)

  • For co-localization studies, include single-stained controls to assess bleed-through

Research has shown that NPAS2 is particularly enriched in reward- and stress-related brain regions such as the striatum and is involved in regulating GABAergic transmission . When designing experiments, consider that knockdown of Npas2 in the ventral striatum has been demonstrated to reduce anxiety-like behaviors, suggesting regional specificity in NPAS2 function.

What are the recommended procedures for ChIP experiments using NPAS2 antibodies?

While FITC-conjugated antibodies are typically not used directly for ChIP, researchers working with NPAS2 often need to perform both immunofluorescence and ChIP studies. Based on published protocols, here are optimized procedures for NPAS2 ChIP experiments:

• ChIP Protocol for NPAS2:

• Primer Design for NPAS2 Target Validation:
  Primers for GABAA receptor subunit genes have been validated for ChIP-qPCR following NPAS2 immunoprecipitation :

  • Gabra1 Forward: 5'-CACACTTGACTGCTGTTTGC-3'

  • Gabra1 Reverse: 5'-GTAGCAGCAGCTATCAACAC-3'

• Timing Considerations:

  • NPAS2 binding to target genes shows circadian oscillation

  • Optimal tissue collection times are ZT4 (day) and ZT16 (night) for comparative analysis

When performing ChIP-Seq analysis, researchers should look for the consensus motif "RRACH" which has been identified as an enriched sequence in NPAS2-binding regions, particularly in the context of m6A modifications .

How should variation in NPAS2 immunofluorescence signal be interpreted and what factors might affect staining intensity?

Several factors can influence NPAS2 staining patterns and intensity when using FITC-conjugated antibodies:

• Biological Variables Affecting NPAS2 Expression:

• Technical Considerations for Signal Optimization:

  • Photobleaching: FITC is susceptible to photobleaching; use antifade mounting media and minimize exposure time

  • Autofluorescence: Brain and kidney tissues show significant green autofluorescence; use Sudan Black B (0.1%) treatment post-staining to reduce background

  • Antibody concentration: Titrate each lot to determine optimal concentration (typically 1:50-1:200)

  • Signal amplification: For low abundance detection, consider using biotin-streptavidin amplification systems

• Interpretation Guidelines:

  • Nuclear NPAS2 signal indicates transcriptionally active protein

  • Cytoplasmic signal may represent newly synthesized or inactive protein

  • Quantification should use nuclear signal intensity for transcription factor activity assessment

  • Normalize to housekeeping proteins when comparing across experimental conditions

Research has shown that NPAS2 expression can be significantly altered by experimental conditions, with both acute and chronic stress increasing levels of Npas2 in the striatum . When analyzing immunofluorescence data, consider these physiological variables as potential sources of variation.

What are the critical considerations for flow cytometric analysis of NPAS2 expression using FITC-conjugated antibodies?

Flow cytometry using NPAS2 Antibody, FITC conjugated requires specific optimization to achieve reliable results:

• Sample Preparation Protocol:

• Controls and Gating Strategy:

  • Fluorescence Minus One (FMO) control is essential for setting NPAS2-FITC gates

  • For macrophage studies, include CD45+F4/80+ gating to identify macrophage populations

  • For compensation, if using multiple fluorophores, single-stained controls are necessary

  • Consider using Npas2 knockdown cells as biological negative controls

• Data Analysis Recommendations:

  • Analyze NPAS2 as median fluorescence intensity (MFI) rather than percent positive

  • Create ratio of NPAS2 MFI to isotype control MFI for standardization across experiments

  • For time-course studies, normalize to Time 0 to track relative changes

  • When studying macrophage polarization, correlate NPAS2 levels with M1 markers (CD86) and M2 markers (CD206)

Research has shown that NPAS2 levels negatively correlate with inflammatory markers in macrophages, with Fto overexpression significantly attenuating NPAS2 expression in kidney tissue of db/db mice . This relationship should be considered when interpreting flow cytometry data of NPAS2 expression in inflammatory contexts.

How can NPAS2 Antibody, FITC conjugated be utilized to investigate NPAS2's regulation of GABAA receptor expression?

Research has established that NPAS2 acts as a transcription factor that binds to and regulates GABAA receptor subunit genes . NPAS2 Antibody, FITC conjugated can be used to investigate this regulatory mechanism through several approaches:

• Co-expression Analysis Protocol:

• Functional Correlation Studies:

  • Following NPAS2 knockdown in the NAc, researchers have observed reduced sensitivity to the GABAa positive allosteric modulator, diazepam

  • Electrophysiological recordings can be combined with immunofluorescence to correlate NPAS2 expression levels with mIPSC amplitude and frequency

  • This approach allows for direct correlation between protein expression and functional outcomes

• Research Applications:

  • Investigation of anxiety disorders and GABAergic dysfunction

  • Studies of circadian regulation of inhibitory neurotransmission

  • Research on alcohol use disorders and GABAergic system alterations

For experimental design, consider that knockdown of Npas2 reduces Gabra1 expression and response to diazepam in the ventral striatum . This suggests that NPAS2 is a critical regulator of GABAergic signaling, with potential implications for anxiety and stress-related disorders.

What protocols are recommended for studying the relationship between NPAS2 and m6A RNA modification?

Recent research has identified NPAS2 as a target of m6A methylation in the context of macrophage activation and diabetic nephropathy . NPAS2 Antibody, FITC conjugated can be used alongside m6A detection methods to investigate this relationship:

• m6A-NPAS2 Co-localization Protocol:

• Combined MeRIP and Immunofluorescence:

  • Perform m6A RNA immunoprecipitation (MeRIP) on one sample set

  • Conduct NPAS2 immunofluorescence on parallel samples

  • Correlate m6A enrichment of Npas2 mRNA with NPAS2 protein levels

  • This approach allows for direct correlation between epitranscriptomic modification and protein expression

• Experimental Considerations:

  • NPAS2 m6A modification occurs within a consensus motif "RRACH"

  • Fto demethylase regulates NPAS2 m6A levels and stability

  • m6A modifications are predominantly mediated through Prrc2a-dependent mechanisms

Research has shown that in diabetic nephropathy, demethylase Fto exhibits low expression and reduces the m6A modification level of Npas2 in macrophages through a Prrc2a-dependent mechanism, decreasing its stability . This process mediates inflammation and glycolysis in M1 macrophage activation by regulating the Hif-1α signaling pathway.