NAP1L4 Antibody

What is NAP1L4 and why is it important in cellular research?

NAP1L4 (Nucleosome Assembly Protein 1-Like 4) is a member of the nucleosome assembly protein family, also known as hNAP2 or NAP2. It has a calculated molecular weight of approximately 43 kDa but is typically observed at 52 kDa in western blots . NAP1L4 plays critical roles in nucleosome assembly and chromatin structure regulation, making it an important target in epigenetic research. Recent studies have revealed its involvement in regulating gene expression through super-enhancer modification, particularly in pulmonary hypertension pathogenesis where it affects cellular proliferation through glycolysis regulation .

What are the most common applications for NAP1L4 antibodies?

NAP1L4 antibodies are widely used in several experimental techniques:

• Western Blot (WB): Typically used at dilutions of 1:1000-1:4000

• Immunoprecipitation (IP): Effective at 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Immunohistochemistry (IHC): Usually performed at dilutions of 1:20-1:200

• Immunofluorescence (IF): Validated in multiple studies

• RNA Immunoprecipitation (RIP): Used to study RNA-protein interactions

The antibody has been validated in multiple cell lines including HeLa, HEK-293, A431, and various mouse tissues such as liver and testis .

What specimen types can be analyzed using NAP1L4 antibodies?

NAP1L4 antibodies have been validated for use with:

• Human cell lines (HeLa, 293T, Jurkat)

• Mouse cell lines (TCMK-1, NIH3T3)

• Human tissue samples (liver tissue in IHC)

• Mouse tissue samples (liver, testis)

• Rat samples

For optimal results in tissue samples, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can be used as an alternative .

How does NAP1L4 interact with circular RNA (circNAP1L4) in pathological conditions?

Recent research has uncovered a fascinating regulatory mechanism involving NAP1L4 and its circular RNA derivative (circNAP1L4). In pulmonary hypertension, circNAP1L4 is downregulated in hypoxic human pulmonary artery smooth muscle cells (HPASMCs) and in plasma of pulmonary hypertension patients .

Mechanistically, circNAP1L4 directly binds to its host protein NAP1L4 in the cytoplasm. When circNAP1L4 levels decrease (as in hypoxic conditions), more NAP1L4 protein can translocate to the nucleus where it regulates the super-enhancer of hexokinase II (HK II), leading to increased glycolysis and cell proliferation . This creates a sophisticated regulatory loop where a circular RNA controls the function of its host protein.

Experimental evidence for this interaction was obtained using RNA immunoprecipitation (RIP) and fluorescence in situ hybridization (FISH) techniques, which demonstrated the colocalization of circNAP1L4 and NAP1L4 protein .

What epigenetic mechanisms are regulated by NAP1L4 and how can they be studied?

NAP1L4 is involved in epigenetic regulation through modifying super-enhancer activities. Specifically, research shows that NAP1L4 regulates super-enhancer modifications of hexokinase II (HK II) by affecting histone marks such as H3K27ac and H3K4me1 .

To study these epigenetic mechanisms:

• Chromatin immunoprecipitation (ChIP) assays using antibodies against H3K27ac, H3K4me1, and NAP1L4 can identify binding sites and regulatory regions

• Combine with RNA-seq or ChIP-seq to map genome-wide changes in gene expression or histone modifications

• Use cellular fractionation to track NAP1L4 nuclear translocation under different conditions

• Employ antibodies like anti-NAP1L4 (16018-1-AP) that have been validated in multiple studies examining nuclear-cytoplasmic distribution

The super-enhancer modification of HK II by NAP1L4 represents an important mechanism by which metabolic reprogramming (increased glycolysis) drives cell proliferation in pathological conditions .

How can splicing factors affect the balance between circNAP1L4 and linear NAP1L4?

Research has identified pre-mRNA-processing-splicing Factor 8 (PRP8) as a key regulator of the production ratio between circNAP1L4 and linear NAP1L4 . This represents an important post-transcriptional regulatory mechanism.

To investigate this relationship:

• Use siRNA-mediated knockdown of PRP8 to observe changes in circNAP1L4 and linear NAP1L4 expression

• Employ qRT-PCR with divergent primers to specifically amplify circNAP1L4

• Use convergent primers to detect linear NAP1L4 mRNA

• Western blot analysis with anti-NAP1L4 antibodies to assess protein levels

• Combine with cellular fractionation to examine subcellular distribution changes

Understanding this regulatory mechanism is crucial as it determines the balance between circRNA and its host gene, ultimately affecting downstream pathways in conditions like pulmonary hypertension .

What are the optimal conditions for using NAP1L4 antibodies in Western blot applications?

For optimal Western blot results with NAP1L4 antibodies:

• Sample preparation: Use NETN lysis buffer for cell lysis as validated in published protocols

• Protein amount: Load approximately 50 μg of whole cell lysate per lane

• Gel type: 4-20% SDS-PAGE gels have been validated for NAP1L4 detection

• Antibody dilution:

  • For NBP2-36543: 0.1-0.4 μg/ml

  • For 16018-1-AP: 1:1000-1:4000

• Expected band size: Approximately 52 kDa, though calculated molecular weight is 43 kDa

• Detection method: Chemiluminescence with exposure times ranging from 30 seconds to 3 minutes depending on signal strength

• Validated cell lines: HeLa, 293T, Jurkat, mouse TCMK-1, and mouse NIH3T3 cells

Note that NAP1L4 antibodies have been successfully used to detect the protein in various species including human, mouse, and rat samples .

What protocol modifications are needed for successful immunoprecipitation of NAP1L4?

For effective immunoprecipitation of NAP1L4:

• Lysate preparation:

  • Use NETN lysis buffer for optimal results

  • Prepare 0.5-1.0 mg of cell lysate per IP reaction

• Antibody amount:

  • Use 6 μg of affinity-purified rabbit anti-NAP1L4 antibody per IP reaction

  • For Proteintech 16018-1-AP, use 0.5-4.0 μg antibody for 1.0-3.0 mg protein lysate

• Loading for Western blot detection:

  • Load approximately 20% of IP product for Western blot analysis

  • Use 0.4 μg/ml antibody concentration for blotting the immunoprecipitated NAP1L4

• Controls:

  • Include IgG control to assess non-specific binding

  • Use multiple antibodies (e.g., NBP2-36542 and NBP2-36543) to confirm specificity

This approach has been validated for successful immunoprecipitation of NAP1L4 from 293T cells with subsequent detection by Western blot .

How should NAP1L4 antibodies be used in co-localization studies with circular RNAs?

For co-localization studies of NAP1L4 protein with circNAP1L4:

• RNA FISH protocol:

  • Fix cells in 4% paraformaldehyde

  • Permeabilize with 0.3% Triton X-100

  • Use Cy3-labeled circNAP1L4 probes designed for specific detection of the circular junction

  • Include 18S and U6 probes as internal controls

  • Counterstain nuclei with DAPI (4′,6-diamidino-2-phenylindole)

• Immunofluorescence for NAP1L4 detection:

  • Use validated antibodies like 16018-1-AP that have been cited in IF applications

  • Apply appropriate dilutions as recommended by manufacturers

  • Use fluorophore-conjugated secondary antibodies with wavelengths distinct from the RNA FISH probes

• Imaging:

  • Capture images using confocal microscopy for optimal resolution of co-localization

  • Use a live cell workstation such as Leica AF6000 for high-quality imaging

• Controls:

  • Include RNase treatment controls to confirm RNA specificity

  • Use antibody pre-absorption controls to verify protein staining specificity

This combined approach allows visualization of RNA-protein interactions and subcellular localization patterns in both cell culture and tissue sections .

How can researchers validate the specificity of NAP1L4 antibodies in their experimental system?

To ensure antibody specificity for NAP1L4:

• Multiple antibody approach:

  • Compare results from different antibody clones (e.g., NBP2-36543, 16018-1-AP, 27889-1-AP)

  • Look for consistent detection patterns across antibodies

• Genetic validation:

  • Use siRNA knockdown or CRISPR-Cas9 knockout of NAP1L4 to confirm signal specificity

  • Several publications have validated NAP1L4 antibodies in knockdown/knockout systems

• Recombinant protein controls:

  • Use purified NAP1L4 recombinant protein as a positive control

  • The immunogen used for 16018-1-AP was a NAP1L4 fusion protein (Ag8827)

• Cross-species reactivity:

  • Test in multiple species to confirm evolutionary conservation (antibodies have been validated in human, mouse, and rat samples)

• Multiple application validation:

  • Confirm consistent results across different techniques (WB, IP, IHC, IF)

  • For example, 16018-1-AP has been validated in WB, IP, IHC, IF, and RIP applications

Proper validation ensures experimental rigor and reproducibility when working with NAP1L4 antibodies.

What factors might affect NAP1L4 detection in different experimental contexts?

Several factors can influence NAP1L4 detection:

• Subcellular localization changes:

  • NAP1L4 shuttles between cytoplasm and nucleus depending on conditions

  • Hypoxia can alter NAP1L4 localization by affecting its interaction with circNAP1L4

  • Cellular fractionation may be necessary to track these changes

• Post-translational modifications:

  • NAP1L4 function may be regulated by modifications affecting antibody recognition

  • Different antibodies may have varying sensitivity to modified forms

• Extraction methods:

  • NETN lysis buffer has been validated for effective NAP1L4 extraction

  • Alternative buffers might affect solubility and detection

• Sample type variations:

  • Different tissue types may require specific antigen retrieval methods

  • For IHC, TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 is an alternative

• Expression level variations:

  • NAP1L4 expression may vary under different physiological or pathological conditions

  • Appropriate positive controls should be included based on known expression patterns

Understanding these factors allows researchers to optimize experimental conditions for reliable NAP1L4 detection.

How should researchers interpret apparent molecular weight differences in NAP1L4 Western blots?

While the calculated molecular weight of NAP1L4 is approximately 43 kDa (for a 375 amino acid protein), it is typically observed at 52 kDa in Western blots . This discrepancy warrants careful interpretation:

• Post-translational modifications:

  • Phosphorylation, glycosylation, or other modifications can increase apparent molecular weight

  • Research has shown that NAP1L4 can be phosphorylated under certain conditions

• Isoform detection:

  • Multiple isoforms of NAP1L4 may exist (e.g., NAP1L4b is mentioned in some literature)

  • Different antibodies may have varying affinities for specific isoforms

• Validation approaches:

  • Compare observed molecular weight across multiple antibodies

  • Use recombinant protein standards with known molecular weights

  • Include positive control samples with validated NAP1L4 detection

• Gel system considerations:

  • Different gel systems (e.g., Tris-glycine vs. Bis-Tris) may affect migration patterns

  • 4-20% gradient gels have been validated for NAP1L4 detection

The consistent observation of NAP1L4 at 52 kDa across multiple studies suggests this is the typical migration pattern for the native protein, despite the lower calculated molecular weight.

How can NAP1L4 antibodies be used to study the role of NAP1L4 in disease mechanisms?

NAP1L4 antibodies have proven valuable in investigating disease mechanisms:

• Pulmonary hypertension research:

  • Anti-NAP1L4 antibodies have been used to track NAP1L4 involvement in pulmonary vascular remodeling

  • Combined with techniques like ChIP to study super-enhancer regulation of glycolysis genes

• Cell proliferation studies:

  • NAP1L4 influences proliferation through epigenetic regulation

  • Antibodies can be used with proliferation markers (PCNA, cyclin A, cyclin D) to study correlations

• Translational research approaches:

  • Monitor NAP1L4 levels in patient samples (plasma, tissue biopsies)

  • Correlate with disease progression or treatment response

  • Validated in human samples using antibodies like 16018-1-AP

• Therapeutic target validation:

  • Use antibodies to validate NAP1L4 as a potential therapeutic target

  • Monitor changes in NAP1L4 levels or localization following experimental interventions

The demonstrated role of NAP1L4 in regulating super-enhancers and cellular proliferation suggests potential involvement in multiple disease contexts beyond pulmonary hypertension.

What are the considerations for studying NAP1L4 protein-circular RNA interactions?

The interaction between NAP1L4 protein and its circular RNA derivative (circNAP1L4) represents a novel regulatory mechanism requiring specialized experimental approaches:

• RNA-protein interaction methods:

  • RNA immunoprecipitation (RIP) using anti-NAP1L4 antibodies (e.g., 16018-1-AP validated for RIP)

  • RNA pull-down assays using biotinylated circNAP1L4 probes

  • CLIP-seq (cross-linking immunoprecipitation with sequencing) for genome-wide interaction mapping

• Co-localization studies:

  • Combined FISH (for circNAP1L4) and immunofluorescence (for NAP1L4 protein)

  • Subcellular fractionation to track localization changes under different conditions

  • Use Cy3-labeled circNAP1L4 probes designed to target the circular junction

• Functional validation:

  • Overexpression or knockdown of circNAP1L4 to observe effects on NAP1L4 localization

  • Site-directed mutagenesis to identify critical interaction domains

  • Downstream pathway analysis (e.g., glycolysis enzyme activity, super-enhancer regulation)

• Physiological relevance:

  • Study interactions under various conditions (normoxia vs. hypoxia)

  • Compare interaction patterns in normal vs. disease states

  • Investigate if the interaction is conserved across different cell types and species