NAP1L5 Antibody

What is NAP1L5 and what is its cellular function?

NAP1L5 belongs to the NAP1L protein family and functions as a histone chaperone. Unlike other NAP1-like proteins that are ubiquitously expressed, NAP1L5 has distinct expression patterns and functions. It was first identified as an imprinted gene in human liver malignancy and later found to be hypomethylated in congenital heart diseases . Recent research has revealed that NAP1L5 promotes nucleolar hypertrophy during cardiomyocyte hypertrophy by regulating translation activation. In neurological contexts, NAP1L5 has been found to regulate APP metabolism and Tau phosphorylation through the GSK3B/Wnt/β-Catenin signaling pathway, suggesting a neuroprotective role in Alzheimer's disease models .

What are the structural characteristics of NAP1L5?

NAP1L5 is a relatively small protein with 182 amino acids and a calculated molecular weight of approximately 20 kDa . Unlike many nuclear proteins, NAP1L5 (formerly known as DRLM) lacks a nuclear localization signal and is primarily distributed in the cytoplasm rather than the nucleus . This cytosolic localization is important for researchers to consider when designing experiments and selecting cellular fractionation methods. The protein's GenBank Accession Number is BC104883 and its UNIPROT ID is Q96NT1, which researchers can reference for sequence information and known modifications .

What types of NAP1L5 antibodies are available for research?

Currently, commercially available NAP1L5 antibodies include polyclonal rabbit antibodies validated for ELISA applications with human samples . For example, Proteintech offers a rabbit polyclonal antibody (18141-1-AP) that has been affinity-purified against a NAP1L5 fusion protein immunogen . This antibody is provided in liquid form in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, and requires storage at -20°C . When selecting an antibody, researchers should consider the specific applications they need (immunofluorescence, Western blotting, immunoprecipitation) and validate cross-reactivity with their species of interest.

How should NAP1L5 antibodies be optimized for immunofluorescence studies?

For immunofluorescence detection of NAP1L5, researchers should consider its cytosolic localization. Based on published studies, NAP1L5 protein has been successfully visualized in the hippocampus CA1 region, where it shows cytosolic distribution and partial co-localization with alpha-smooth muscle actin (α-SMA) . For optimal results, tissue sections should be appropriately fixed and permeabilized to ensure antibody access to cytosolic proteins. A working dilution must be empirically determined, but starting with manufacturer recommendations is advised. Counterstaining with markers such as NeuN (for neurons) or α-SMA can provide valuable context about cell-specific expression patterns, as demonstrated in studies with APP/PS1 mouse models .

What protocols yield the best results for NAP1L5 Western blotting?

For Western blotting applications, researchers should optimize protein extraction protocols to efficiently isolate cytosolic proteins where NAP1L5 is primarily located. Based on published results, standard SDS-PAGE with transfer to PVDF membranes has successfully detected NAP1L5 at its expected molecular weight of 20 kDa . When comparing NAP1L5 expression between different samples (e.g., control vs. disease models), normalization to appropriate housekeeping proteins is essential. Researchers have successfully used this approach to demonstrate decreased NAP1L5 protein levels in N2a-APP695sw cells compared to N2a cells, providing quantifiable evidence of NAP1L5 downregulation in AD models .

How can researchers validate NAP1L5 antibody specificity?

Validation of antibody specificity is crucial for reliable results. Recommended approaches include: 1) Using positive and negative control samples (e.g., comparing wild-type tissue with NAP1L5 knockout models or using cell lines with known expression levels); 2) Performing peptide competition assays where pre-incubation of the antibody with its antigenic peptide should abolish specific signals; 3) Validating antibody specificity in overexpression systems, as demonstrated in studies using lentivirus-mediated NAP1L5-overexpressing N2a-APP695sw cells to confirm antibody specificity . Additionally, researchers should verify that the detected band corresponds to the expected molecular weight of 20 kDa for NAP1L5 .

How is NAP1L5 expression altered in Alzheimer's disease?

Bioinformatic analysis of transcriptome data from the Gene Chip Public Database (GEO) and GSE37263 microarray data has revealed that NAP1L5 is significantly downregulated in the brain tissues of AD patients compared to controls . This finding has been experimentally validated at both mRNA and protein levels in various models. In the N2a-APP695sw cell model of AD, NAP1L5 mRNA and protein levels are significantly reduced compared to N2a control cells . Immunofluorescence studies have further confirmed markedly reduced NAP1L5 expression in the hippocampus CA1 region of APP/PS1 mouse models . This consistent downregulation across different experimental systems and human samples suggests a potential role for NAP1L5 in AD pathogenesis.

What methodologies are recommended for studying NAP1L5 in AD models?

For comprehensive investigation of NAP1L5 in AD models, researchers should employ a multi-faceted approach. First, expression analysis using RT-qPCR for mRNA and Western blotting for protein levels provides quantitative measures of NAP1L5 expression changes . Cell models such as N2a-APP695sw cells offer a controlled system for mechanistic studies, while transgenic models like APP/PS1 mice allow for in vivo validation . To investigate functional effects, lentivirus-mediated overexpression systems have proven effective in restoring NAP1L5 levels and studying subsequent effects on AD pathology markers . Immunofluorescence co-localization studies with neuronal markers (NeuN) or vascular markers (α-SMA) help determine cell type-specific expression and alterations .

How does NAP1L5 interact with APP metabolism and Tau phosphorylation?

Experimental evidence indicates that NAP1L5 plays a regulatory role in APP metabolism and Tau phosphorylation through the GSK3B/Wnt/β-Catenin signaling pathway . In N2a-APP695sw cells, NAP1L5 overexpression significantly reduces APP protein levels, which are typically elevated in these AD model cells compared to normal N2a cells . Additionally, NAP1L5 overexpression decreases the production of sAPPβ and Aβ, which are critical metabolites in the amyloidogenic pathway . NAP1L5 also inhibits BACE1 expression, a key β-secretase enzyme that mediates the first stage of APP proteolysis in the amyloid pathway . Together, these findings suggest that NAP1L5 has neuroprotective effects by inhibiting the amyloidogenic processing of APP, possibly through its interactions with the GSK3B/Wnt/β-Catenin pathway .

How can apparent contradictions in NAP1L5 function across different disease models be reconciled?

NAP1L5 has been studied in different disease contexts, including Alzheimer's disease, liver malignancy, and congenital heart diseases, with potentially different functional implications . These apparent contradictions might be reconciled through several approaches: 1) Investigating tissue-specific protein interactions and signaling pathways that might direct NAP1L5 toward different functions; 2) Examining post-translational modifications that could alter NAP1L5 function in disease-specific contexts; 3) Determining if different NAP1L5 isoforms exist and predominate in different tissues; and 4) Conducting comparative studies using the same antibodies and methodologies across different disease models to eliminate technical variations. A systems biology approach that integrates proteomic, transcriptomic, and functional data could help build a unified model of NAP1L5 function across different physiological and pathological contexts.

What novel applications exist for NAP1L5 antibodies in cutting-edge research?

Emerging research suggests several novel applications for NAP1L5 antibodies. First, they could serve as tools for developing diagnostic biomarkers for AD, given the consistent downregulation of NAP1L5 in AD brain tissues . Second, NAP1L5 antibodies could be used in high-throughput screens to identify compounds that restore NAP1L5 expression or function, potentially leading to novel therapeutic approaches for AD . Third, combining NAP1L5 antibodies with proximity ligation assays could help identify novel protein interaction partners, enhancing our understanding of NAP1L5's role in the GSK3B/Wnt/β-Catenin pathway . Fourth, developing antibodies specific to post-translational modifications of NAP1L5 could reveal regulatory mechanisms that might be targeted therapeutically. Finally, NAP1L5 antibodies could be valuable tools in studying the relationship between NAP1L5 and AQP1, as these proteins were found to be negatively correlated in neurodegenerative disease datasets .

What controls are essential for NAP1L5 antibody-based experiments?

Several controls are critical for ensuring reliable results in NAP1L5 antibody experiments. For Western blotting: 1) Positive controls using tissues/cells known to express NAP1L5 (e.g., specific brain regions); 2) Negative controls using tissues with minimal NAP1L5 expression or NAP1L5 knockdown samples; 3) Loading controls with appropriate housekeeping proteins for quantification; and 4) Molecular weight markers to confirm the expected 20 kDa band for NAP1L5 . For immunofluorescence: 1) Primary antibody omission control; 2) Secondary antibody-only control to assess non-specific binding; 3) Positive control tissues (e.g., wild-type hippocampus); and 4) Negative controls (e.g., brain regions with minimal NAP1L5 expression) . For functional studies, comparing wild-type cells to those with manipulated NAP1L5 levels (overexpression or knockdown) is essential for attributing observed effects specifically to NAP1L5 .

How should researchers account for cytosolic localization of NAP1L5 in experimental design?

The cytosolic localization of NAP1L5 has important implications for experimental design . For protein extraction, cytosolic fractionation protocols should be optimized to efficiently isolate NAP1L5 without nuclear contamination. Subcellular fractionation studies should include markers for cytosolic (e.g., GAPDH) and nuclear (e.g., Histone H3) compartments to confirm proper separation. For immunofluorescence, permeabilization conditions should be optimized for cytosolic proteins, and co-staining with compartment-specific markers helps confirm the cytosolic localization . When studying protein-protein interactions, researchers should focus on cytosolic binding partners rather than nuclear proteins. Additionally, when designing expression vectors for NAP1L5 overexpression studies, avoid adding tags that might inadvertently alter the protein's subcellular localization, as this could affect its functional interactions.