NPAS2 Antibody, HRP conjugated

What is NPAS2 and why is it significant in research?

NPAS2 is a member of the basic helix-loop-helix (bHLH)-PAS family of transcription factors that functions as a core component of the circadian clock. It plays a regulatory role in the acquisition of specific types of memory and functions as part of the molecular clock operative in the mammalian forebrain. NPAS2 acts as a transcriptional activator within the circadian system, regulating various physiological processes through the generation of approximately 24-hour circadian rhythms in gene expression, which translate into rhythms in metabolism and behavior . Research on NPAS2 is significant for understanding the fundamental mechanisms of circadian biology and its implications in memory formation, metabolic regulation, and neurological functions.

What applications are suitable for NPAS2 antibody, HRP conjugated?

NPAS2 antibody with HRP conjugation is primarily suitable for:

• Western Blot (WB) - Detection of NPAS2 protein at approximately 92 kDa molecular weight

• ELISA - Quantitative measurement of NPAS2 protein levels

The HRP conjugation eliminates the need for secondary antibody incubation, reducing experimental time and potential background issues. For Western blot applications, the recommended working concentration is 1 μg/mL, while for ELISA applications, a dilution of 1:1000 to 1:62500 is typically recommended .

What are the optimal storage conditions for maintaining antibody activity?

To maintain optimal activity of NPAS2 antibody, HRP conjugated:

• Store at -20°C for long-term storage

• Aliquot into multiple vials to avoid repeated freeze-thaw cycles

• For short periods (days), 4°C storage is acceptable

• If antibody is lyophilized, reconstitute by adding 50 μL of distilled water to achieve a final concentration of 1 mg/mL

• After reconstitution, store in buffer containing glycerol (typically 50%) to prevent freezing damage

The antibody is typically provided in a storage buffer containing PBS (pH 7.2-7.4), often with stabilizers like BSA (1%), Proclin300 (0.03%), and glycerol (50%) . Multiple freeze-thaw cycles significantly reduce antibody activity and should be avoided through proper aliquoting.

What is the reactivity profile of commercially available NPAS2 antibodies?

Most commercially available NPAS2 antibodies, including HRP-conjugated versions, demonstrate reactivity against:

The reactivity is determined by the conservation of the epitope region across species. Most antibodies are generated against synthetic peptides derived from human NPAS2, particularly the region spanning amino acids 631-730/824 or the N-terminal region (amino acids 1-30), enabling cross-reactivity with mouse and rat homologs due to high sequence homology .

How does the m6A modification affect NPAS2 expression and what methods can detect this?

Recent research has revealed that NPAS2 undergoes m6A RNA modification, which significantly impacts its stability and expression. The m6A demethylase Fto reduces the m6A modification level of NPAS2 in macrophages through a Prrc2a-dependent mechanism, decreasing its stability. This modification has implications for macrophage activation and inflammatory responses .

To investigate this modification:

• Methylated RNA immunoprecipitation (MeRIP) combined with qPCR can detect changes in m6A levels on NPAS2 mRNA

• RNA stability assays using actinomycin D treatment followed by time-course RT-qPCR can measure NPAS2 mRNA stability

• RNA immunoprecipitation (RIP) analysis can confirm the binding of m6A readers like Prrc2a to NPAS2 mRNA

• Luciferase reporters containing wild-type or mutant NPAS2 constructs can assess the functional impact of m6A modifications

Researchers investigating NPAS2 regulation should consider these epigenetic modifications, as they may significantly impact experimental outcomes and interpretations, particularly in inflammatory and metabolic research contexts .

What are the critical considerations for optimizing NPAS2 western blot detection using HRP-conjugated antibodies?

When optimizing western blot detection of NPAS2 using HRP-conjugated antibodies, consider:

• Sample preparation:

  • Include protease inhibitors to prevent NPAS2 degradation

  • Consider nuclear extraction protocols as NPAS2 is a nuclear transcription factor

  • Use positive controls like fetal heart tissue lysate to validate detection

• Electrophoresis conditions:

  • NPAS2 has a predicted molecular weight of 92 kDa

  • Use 8-10% SDS-PAGE gels for optimal separation

  • Extended transfer times (60-90 minutes) may be necessary for complete transfer

• Antibody concentrations:

  • For HRP-conjugated NPAS2 antibodies, use at 1 μg/mL concentration

  • Adjust concentration based on signal-to-noise ratio

• Exposure optimization:

  • Start with short exposures (30 seconds) and increase if necessary

  • HRP signal develops rapidly and may saturate, so monitor carefully

  • Consider using chemiluminescence enhancers for weak signals

• Verification strategies:

  • Use multiple antibodies targeting different epitopes to confirm specificity

  • Consider knockdown/knockout controls to validate band identity

How can researchers effectively design experiments to study NPAS2's role in circadian regulation using HRP-conjugated antibodies?

To design effective experiments investigating NPAS2's role in circadian regulation:

• Temporal sampling strategy:

  • Collect samples at multiple time points across 24-hour cycles (minimum 4-6 timepoints)

  • For initial screening, collect samples every 4 hours

  • For detailed analysis, collect samples every 2 hours during critical circadian transitions

• Antibody selection:

  • Use HRP-conjugated NPAS2 antibodies for direct detection in time-course studies

  • Consider using antibodies targeting specific post-translational modifications for mechanistic studies

• Analytical approaches:

  • Combine protein detection (Western blot) with mRNA analysis (qPCR)

  • Correlate NPAS2 protein levels with expression of known target genes

  • Consider chromatin immunoprecipitation (ChIP) to assess NPAS2 binding to circadian gene promoters

• Synchronization protocols:

  • For cell culture: Use serum shock or dexamethasone treatment to synchronize cellular clocks

  • For tissue samples: Harvest at consistent times relative to entrainment cues (light/dark cycles)

• Data analysis:

  • Apply cosinor analysis to quantify rhythmic parameters (amplitude, period, phase)

  • Use appropriate statistical tests that account for circadian variations

What are the mechanisms through which NPAS2 influences macrophage inflammation and glycolysis?

NPAS2 plays a significant role in regulating macrophage inflammation and glycolysis through several interconnected mechanisms:

• Transcriptional regulation:

  • NPAS2 positively regulates the expression of pro-inflammatory cytokines including IL-1β, TNF-α, and MCP-1

  • It also upregulates glycolytic enzymes HK1 and PFKFB3, enhancing glycolytic flux

• HIF-1α pathway connection:

  • NPAS2 appears to function through HIF-1α signaling pathway-dependent mechanisms

  • Downregulation of NPAS2 reduces expression of glycolytic enzymes and inflammatory markers

• m6A modification influence:

  • NPAS2 expression and stability are regulated by m6A RNA modifications

  • The m6A demethylase Fto reduces NPAS2 stability through a Prrc2a-dependent mechanism

  • This regulatory axis affects macrophage activation and inflammatory responses

• Physiological impact:

  • In diabetic nephropathy models, silencing NPAS2 effectively reduces kidney damage

  • NPAS2 suppression inhibits basement membrane thickening and foot process fusion

  • Decreased NPAS2 expression reduces F4/80+ inflammatory cell infiltration

Experimental approaches using HRP-conjugated NPAS2 antibodies can help investigate these pathways by tracking NPAS2 protein levels in response to inflammatory stimuli, metabolic alterations, or pharmacological interventions .

What methodological approaches can distinguish between transcriptional and post-translational regulation of NPAS2?

To distinguish between transcriptional and post-translational regulation of NPAS2:

• Transcriptional regulation analysis:

  • Quantitative RT-PCR to measure NPAS2 mRNA levels

  • Nuclear run-on assays to measure nascent transcription rates

  • Reporter gene assays using NPAS2 promoter constructs

  • ChIP assays to identify transcription factors binding to NPAS2 promoter

• Post-translational modification analysis:

  • Western blot with phospho-specific or other PTM-specific antibodies

  • Immunoprecipitation followed by mass spectrometry to identify modifications

  • Pulse-chase experiments to measure protein turnover rates

  • Proteasome inhibitors to assess degradation pathways

• Integrated approaches:

  • Compare protein levels (using HRP-conjugated antibodies) with mRNA levels

  • If changes occur at protein level without corresponding mRNA changes, post-translational regulation is likely

  • If both change correspondingly, transcriptional regulation is more likely

• Temporal considerations:

  • Conduct time-course experiments to distinguish immediate vs. delayed effects

  • Rapid changes (minutes to hours) often involve post-translational mechanisms

  • Slower changes (hours to days) may involve transcriptional regulation

What controls should be included when using NPAS2 antibody, HRP conjugated in immunological assays?

When using NPAS2 antibody, HRP conjugated, the following controls should be included:

• Positive controls:

  • Fetal heart tissue lysate (validated for NPAS2 detection)

  • Cells or tissues known to express high levels of NPAS2 (e.g., brain tissues, particularly forebrain)

  • Recombinant NPAS2 protein (if available)

• Negative controls:

  • NPAS2 knockout or knockdown samples (if available)

  • Tissues known not to express NPAS2

  • Primary antibody omission control

• Specificity controls:

  • Pre-absorption with immunizing peptide to confirm epitope specificity

  • Use of multiple antibodies targeting different NPAS2 epitopes

  • Secondary antibody-only control (though less relevant for direct HRP conjugates)

• Loading and transfer controls:

  • Housekeeping proteins (β-actin, GAPDH) for total protein normalization

  • Ponceau S staining to confirm protein transfer

  • Normalization to total protein using stain-free technology

• Technical controls:

  • Dilution series to confirm linear range of detection

  • Replicate samples to assess technical variability

How can researchers effectively validate antibody specificity for NPAS2 detection in different experimental systems?

To validate NPAS2 antibody specificity across experimental systems:

• Genetic validation approaches:

  • NPAS2 knockdown/knockout verification: Compare antibody signals between wild-type and NPAS2-deficient samples

  • Overexpression validation: Test antibody performance in systems with controlled NPAS2 overexpression

  • Heterologous expression: Express NPAS2 in cells that normally lack expression

• Biochemical validation methods:

  • Immunoprecipitation followed by mass spectrometry to confirm captured protein identity

  • Epitope competition assays using the immunizing peptide

  • Western blot molecular weight verification (NPAS2 ~92 kDa)

  • Detection of expected band pattern changes in different physiological conditions

• Cross-platform validation:

  • Compare results between different detection methods (WB, ELISA, IHC)

  • Use multiple antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression data

• Species-specific considerations:

  • When using the antibody across species, compare sequence homology in the epitope region

  • Validate in each species independently

  • Be aware that most NPAS2 antibodies are raised against human epitopes but show cross-reactivity with mouse and rat

What are common issues when using NPAS2 antibody, HRP conjugated in western blot and how can they be resolved?

Common issues and their solutions when using HRP-conjugated NPAS2 antibodies:

For persistent issues, consider:

• Comparing the performance of unconjugated antibodies with secondary detection

• Testing different extraction methods to preserve NPAS2 integrity

• Using fresh HRP substrate solutions

• Optimizing incubation times and temperatures

How can researchers address variability in NPAS2 detection across different tissue and cell types?

To address variability in NPAS2 detection across different samples:

• Sample-specific optimization:

  • Adjust protein extraction protocols for each tissue/cell type

  • For brain tissues, use specialized nuclear extraction protocols

  • For tissues with high protease activity, increase protease inhibitor concentrations

• Expression level considerations:

  • NPAS2 expression varies significantly by tissue and circadian time

  • Brain tissues (particularly forebrain) typically show highest expression

  • For low-expressing tissues, increase protein loading and extend exposure times

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Some tissues may require tissue-specific blockers to reduce background

  • Optimize blocking time (1-2 hours) and temperature

• Detection enhancement strategies:

  • For tissues with low NPAS2 expression, use high-sensitivity ECL substrates

  • Consider signal amplification systems for very low abundance

  • Increase antibody incubation time (overnight at 4°C)

• Normalization approaches:

  • Use tissue-specific housekeeping controls

  • Consider total protein normalization instead of single reference proteins

  • Include inter-tissue calibration samples when comparing across tissues

How should researchers interpret changes in NPAS2 levels in the context of m6A modification research?

When interpreting NPAS2 level changes in m6A modification research:

• Distinguishing direct vs. indirect effects:

  • Determine if changes in NPAS2 protein levels correlate with mRNA levels

  • Assess m6A modification levels on NPAS2 mRNA using MeRIP-qPCR

  • Measure NPAS2 mRNA stability with and without m6A pathway manipulation

• Considering reader protein interactions:

  • Evaluate the role of m6A reader proteins, particularly Prrc2a

  • Perform RNA immunoprecipitation to assess Prrc2a binding to NPAS2 mRNA

  • Consider knockdown of Prrc2a to determine its impact on NPAS2 levels

• Temporal dynamics:

  • Examine time-course changes in NPAS2 levels after m6A pathway manipulation

  • Consider that m6A effects may have different kinetics than transcriptional regulation

  • Rapid changes may indicate direct m6A-mediated regulation

• Functional validation approaches:

  • Assess downstream effects on inflammatory markers and glycolytic enzymes

  • Correlate NPAS2 levels with Il-1β, Tnf-α, Mcp-1, Hk1, and Pfkfb3 expression

  • Use luciferase reporter assays with wild-type vs. m6A-mutant NPAS2 constructs

• Methodological considerations:

  • When using HRP-conjugated antibodies, ensure equal loading and transfer

  • Include appropriate controls for m6A pathway manipulations

  • Consider using both Western blot (protein) and qPCR (mRNA) for comprehensive analysis