NPAS4 Antibody, HRP conjugated

What is NPAS4 and why is it important in research?

NPAS4 is an activity-dependent transcription factor expressed in various tissues, particularly neurons and pancreatic β-cells. In neurons, it regulates inhibitory synapse development while in β-cells, it functions as a cytoprotective factor that improves cellular efficiency under stress conditions . NPAS4 is rapidly induced following depolarization in a calcium-dependent manner, making it an important marker of cellular activity and stress response . Its ability to bind enhancer regions of immediate early genes like Fos and Per1 positions it as a key regulator of transcriptional networks .

What is the molecular weight and structure of NPAS4?

NPAS4 is a basic helix-loop-helix PAS (bHLH-PAS) domain transcription factor with a molecular weight of approximately 100 kDa. The protein contains DNA-binding domains that allow it to interact with specific enhancer regions of target genes . When using HRP-conjugated antibodies for detection, researchers should expect to observe bands at around 100 kDa in Western blot applications, with the exact size potentially varying slightly due to post-translational modifications.

What is the typical expression pattern of NPAS4 during development?

Developmental expression of NPAS4 shows tissue-specific patterns. In pancreatic β-cells, NPAS4 is dramatically induced just prior to birth and continues to be expressed in adult islets . Real-time PCR analysis demonstrates this developmental timing is crucial for normal β-cell function. Understanding this expression pattern is essential when designing experiments with NPAS4 antibodies across developmental timepoints.

What are the optimal conditions for NPAS4 detection in Western blotting using HRP-conjugated antibodies?

For optimal Western blot detection of NPAS4 using HRP-conjugated antibodies, consider the following parameters:

The timing of sample collection is particularly critical as NPAS4 expression is dynamic, with protein levels typically peaking 2-4 hours after stimulation in many cell types .

How can researchers validate NPAS4 antibody specificity?

Validation of NPAS4 antibody specificity should include multiple approaches:

• Positive controls: Use tissues or cells known to express NPAS4, such as depolarized neurons or β-cells stimulated with glucose or KCl

• Negative controls: Test in tissues where NPAS4 is not expressed or use NPAS4 knockout models

• Peptide competition assays: Pre-incubation of antibody with immunizing peptide should abolish signal

• Knockdown validation: siRNA or shRNA against NPAS4 should reduce antibody signal proportionally

• Western blot migration pattern: Compare observed band size with expected molecular weight

• Cross-validation: Compare results from antibodies targeting different NPAS4 epitopes

What are the critical parameters for successful immunohistochemistry with HRP-conjugated NPAS4 antibodies?

For immunohistochemical detection of NPAS4:

• Fixation: 4% paraformaldehyde is typically effective

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Permeabilization: 0.1-0.3% Triton X-100

• Blocking: 5-10% normal serum from secondary antibody species

• Antibody dilution: 1:100 to 1:500 for HRP-conjugated primary antibodies

• Incubation: Overnight at 4°C for optimal binding

• Development: DAB substrate with optimization of development time (typically 1-5 minutes)

• Counterstaining: Hematoxylin for nuclear visualization

• Controls: Include primary antibody omission controls

When examining NPAS4 expression in pancreatic islets, immunofluorescence analysis reveals NPAS4 is expressed in both α- and β-cells but not in the exocrine tissue . Significant heterogeneity in staining intensity between islets has been observed, suggesting dynamic regulation.

How can researchers effectively study NPAS4-mediated transcriptional regulation using ChIP assays?

NPAS4 binds to specific enhancer regions of target genes as shown in multiple ChIP-seq datasets . For studying NPAS4-mediated transcriptional regulation:

• Chromatin preparation: Fix cells with 1% formaldehyde for 10 minutes

• Sonication optimization: Aim for fragments of 200-500 bp

• Antibody selection: Use ChIP-validated NPAS4 antibodies (including HRP-conjugated versions for certain detection methods)

• Controls: Include IgG control and positive control regions (known NPAS4 targets)

• Primer design: Target known NPAS4 binding regions in enhancers of genes like Fos, Per1, or Rgs2

• Data normalization: Express results as relative binding percentage compared to input samples

• Integration with other data: Combine with RNA-seq or histone modification ChIP data

Research demonstrates that NPAS4 binds to specific enhancer regions in both neurons and β-cells, with ChIP assays showing direct binding to intron 1 and enhancer regions of target genes like Rgs2 .

What approaches are recommended for studying NPAS4 in cellular stress response?

When investigating NPAS4's role in stress response:

• Cell type selection: Choose appropriate cell types as NPAS4 functions differently in neurons versus β-cells

• Stress paradigm: Select relevant stressors (e.g., thapsigargin for ER stress, palmitate for lipotoxicity)

• Temporal dynamics: Design time-course experiments capturing both early (1-2h) and late (24h) responses

• Functional readouts: Measure stress markers (DDIT3/CHOP), cytoprotective factors (Hspa5, Wfs-1), and apoptosis (TUNEL assay)

• Gain/loss of function: Combine overexpression and knockdown approaches

Research shows NPAS4 is induced by ER stressors like thapsigargin and palmitate in β-cells and provides protection by reducing expression of proapoptotic factors like DDIT3 while increasing cytoprotective factors like Wfs-1 and Hspa5 .

How can researchers analyze NPAS4 regulation of incretin-stimulated insulin secretion?

To study NPAS4's role in modulating incretin-stimulated insulin secretion:

• Experimental system: Use MIN6 cells or isolated islets with adenoviral NPAS4 expression

• Secretion assays: Measure insulin secretion in response to glucose alone versus glucose plus incretin (e.g., exendin-4)

• cAMP measurements: Assess exendin-4-stimulated cAMP production

• Target gene analysis: Measure expression of Rgs2, which negatively regulates incretin-mediated cAMP production

• ChIP analysis: Confirm NPAS4 binding to Rgs2 regulatory regions

Research demonstrates that NPAS4 inhibits incretin-stimulated insulin secretion without significantly affecting glucose-stimulated secretion by inducing Rgs2 expression and reducing cAMP production .

What are common problems encountered when using NPAS4 antibodies and how can they be resolved?

Common issues and solutions include:

How can researchers address the technical challenges in detecting phosphorylated HDAC5 and its relationship with NPAS4?

The relationship between phosphorylated HDAC5 and NPAS4 expression presents several technical challenges:

• Sample preparation: Include phosphatase inhibitors in all buffers to prevent dephosphorylation

• Antibody selection: Use phospho-specific antibodies validated for the specific phosphorylation site

• Controls: Include positive controls (e.g., CGRP-treated samples) that are known to increase HDAC5 phosphorylation

• ChIP assays: Assess HDAC5 binding to NPAS4 enhancer regions before and after treatments that induce phosphorylation

• Functional validation: Use PKD inhibitors (e.g., H89) or Prkd1-siRNA to verify the signaling pathway

Research shows that CGRP treatment decreases HDAC5 binding to the NPAS4 enhancer by approximately 50%, while increasing histone H3 acetylation at the same site . PKD inhibition with H89 or Prkd1-siRNA significantly reduces both phosphorylated HDAC5 and NPAS4 protein levels, confirming the regulatory relationship .

How can researchers resolve contradictory findings regarding NPAS4 function across different cell types?

To address contradictions in NPAS4 research across cell types:

• Side-by-side comparisons: Study multiple cell types under identical experimental conditions

• Context consideration: Account for differential expression of binding partners and cofactors

• Temporal dynamics: Compare expression and function kinetics

• Downstream targets: Identify cell type-specific target genes using ChIP-seq and RNA-seq

• Post-translational modifications: Assess differences in modifications that might affect function

• Signaling context: Consider cell type-specific signaling pathways that might modify NPAS4 activity

Research demonstrates NPAS4 has distinct roles in neurons (regulating inhibitory synapse development) versus β-cells (modulating insulin production and protecting against ER stress) , highlighting the importance of cellular context.

What are promising approaches for studying NPAS4 in vivo that would benefit from HRP-conjugated antibodies?

Future research directions include:

• Conditional knockout models to study cell type-specific functions

• In vivo ChIP-seq to identify physiologically relevant binding sites

• Single-cell approaches to address heterogeneity in NPAS4 expression

• In vivo imaging of NPAS4 expression dynamics during physiological stimulation

• Therapeutic targeting of NPAS4 pathways in diabetes models

The cytoprotective properties of NPAS4 in β-cells make it a potential therapeutic target for type 2 diabetes, as it could reduce ER stress and cell death while maintaining basal cell function .

How might emerging technologies enhance our understanding of NPAS4 function?

Emerging technologies with potential applications include:

• CUT&RUN or CUT&Tag for higher resolution binding profiles

• CRISPR screens to identify functional NPAS4 targets

• Proximity labeling methods (BioID, APEX) to identify NPAS4 interactors

• Chromosome conformation capture techniques to study enhancer-promoter interactions

• Optical control of NPAS4 expression to examine temporal requirements

• Mass spectrometry to identify post-translational modifications

These approaches would complement traditional antibody-based methods and provide deeper insights into NPAS4 biology.