NHP2L1 Antibody

What is NHP2L1 and what cellular functions does it perform?

NHP2L1 (also known as SNU13, High mobility group-like nuclear protein 2 homolog 1, or U4/U6.U5 tri-snRNP 15.5 kDa protein) is a multifunctional protein involved in several critical cellular processes. It serves as part of the small subunit (SSU) processome, which is the first precursor of the small eukaryotic ribosomal subunit. During SSU processome assembly in the nucleolus, NHP2L1 works alongside other ribosome biogenesis factors to generate RNA folding, modifications, rearrangements, and cleavage. Additionally, it participates in pre-mRNA splicing as a component of the spliceosome. NHP2L1 specifically binds to the 5'-stem-loop of U4 snRNA, contributing to spliceosome assembly. The protein undergoes significant conformational changes upon RNA binding, highlighting its dynamic role in RNA processing .

What types of NHP2L1 antibodies are available for research applications?

Research laboratories can utilize several types of NHP2L1 antibodies, including rabbit recombinant monoclonal antibodies (such as EPR11671) and rabbit polyclonal antibodies. These antibodies are available in various formats, including standard formulations and carrier-free versions designed for conjugation to fluorochromes, metal isotopes, oligonucleotides, and enzymes. The carrier-free formats are particularly valuable for antibody labeling, functional assays, cell-based assays, flow-based assays (including mass cytometry), and multiplex imaging applications .

What experimental applications are NHP2L1 antibodies suitable for?

NHP2L1 antibodies have been validated for multiple experimental applications in molecular and cellular biology research. Most commercial antibodies are suitable for Western blot (WB), immunohistochemistry on paraffin-embedded tissues (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF). Some antibodies, particularly the monoclonal variants, have additional validated applications including immunoprecipitation (IP) and intracellular flow cytometry. For Western blotting, these antibodies can typically detect the predicted band size of approximately 14 kDa corresponding to NHP2L1 protein in various human cell lines including HepG2, HeLa, Jurkat, and MCF-7 .

What are the optimal conditions for immunohistochemistry with NHP2L1 antibodies?

For optimal immunohistochemical detection of NHP2L1 in paraffin-embedded tissues, a validated protocol includes heat-mediated antigen retrieval with EDTA buffer at pH 9 before commencing with the IHC staining protocol. The antibody can be used at dilutions ranging from 1/500 to 1/1000, followed by an appropriate secondary detection system such as HRP Polymer for Rabbit IgG and hematoxylin counterstain. This approach has been successfully applied to human lung carcinoma tissue and provides clear visualization of NHP2L1 expression patterns .

How can researchers optimize Western blot protocols for NHP2L1 detection?

For optimal Western blot detection of NHP2L1, researchers should begin with cell lysate preparation from appropriate cell lines (such as HepG2, HeLa, Jurkat, or MCF-7) at a loading concentration of approximately 20 μg per lane. Antibody dilutions of 1/10000 to 1/20000 for primary antibody incubation followed by detection with anti-rabbit IgG peroxidase-conjugated secondary antibody at 1/1000 dilution provide excellent signal-to-noise ratios. Researchers should anticipate a band at approximately 14 kDa, corresponding to the predicted molecular weight of NHP2L1. Optimization of blocking conditions and incubation times may be necessary depending on the specific experimental setup and detection method employed .

How can researchers study the interaction between NHP2L1 and U4 snRNA?

Researchers can investigate the interaction between NHP2L1 and U4 snRNA using time-resolved FRET (TR-FRET). This methodology involves preparing recombinant NHP2L1 protein and synthetic U4 5' stem-loop RNA labeled with appropriate FRET pairs. The assay measures the fluorescence emission ratio (10,000 × 665nm/620nm) to quantify interaction. This approach is particularly valuable for high-throughput screening of compounds that may disrupt the NHP2L1-U4 interaction. When establishing the assay, researchers should include appropriate controls: complete interaction mixture (negative control, 0% inhibition) and partial interaction mixture (positive control, 100% inhibition). The TR-FRET approach has been successfully employed to identify small molecules, such as topotecan, that interfere with this critical spliceosomal interaction .

What approaches can be used to investigate NHP2L1's role in splicing mechanisms?

To investigate NHP2L1's role in splicing mechanisms, researchers can employ multiple complementary approaches. Nuclear magnetic resonance (NMR) spectroscopy using 15N-labeled NHP2L1 allows monitoring of chemical shift perturbations upon interaction with U4 RNA and potential small molecule inhibitors. These experiments should include 2D [15N, 1H] HSQC or TROSY spectra to track conformational changes. Additionally, researchers can use cell-based splicing reporter assays following NHP2L1 knockdown or inhibition of its interaction with U4 (using compounds like topotecan). For in-depth mechanistic studies, reconstituted splicing assays with purified components and NHP2L1 protein variants can help elucidate the precise role of specific protein domains in spliceosome assembly and function .

How can NHP2L1's role in the small subunit processome be studied experimentally?

Investigating NHP2L1's role in the small subunit processome requires specialized approaches focused on ribosome biogenesis. Researchers can employ RNA immunoprecipitation (RIP) using NHP2L1 antibodies to isolate and identify associated pre-rRNAs and other RNA species. Chromatin immunoprecipitation (ChIP) can also reveal NHP2L1's association with active ribosomal DNA loci. For functional studies, CRISPR/Cas9-mediated gene editing to create conditional NHP2L1 knockout or domain-specific mutants, followed by ribosome profiling and pre-rRNA processing analysis by Northern blotting, can provide insights into how NHP2L1 contributes to small subunit assembly. Additionally, proximity labeling approaches (BioID or APEX) with NHP2L1 as the bait protein can identify novel interaction partners within the processome complex .

What are common issues in Western blotting with NHP2L1 antibodies and how can they be resolved?

When performing Western blotting with NHP2L1 antibodies, researchers may encounter several challenges. Non-specific bands can appear, particularly in the 15-20 kDa range. To address this, optimize blocking conditions (try 5% BSA instead of milk), increase antibody dilution (1/20000 rather than 1/10000), and implement more stringent washing procedures. Another common issue is weak or absent signal for the 14 kDa NHP2L1 protein, which may result from protein degradation during sample preparation. Use fresh protease inhibitors and maintain samples at 4°C throughout processing. If detecting NHP2L1 in nuclear extracts, ensure complete nuclear lysis, as the protein's tight association with nuclear structures may limit extraction efficiency. Finally, if working with tissue samples, optimization of extraction buffers may be necessary to efficiently solubilize NHP2L1 from its native complexes within the spliceosome and SSU processome .

How can researchers distinguish between specific and non-specific staining in immunohistochemistry?

Distinguishing between specific and non-specific staining in NHP2L1 immunohistochemistry requires rigorous controls and careful protocol optimization. First, always include a no-primary-antibody control to assess background from the detection system. Second, use tissues known to express high levels of NHP2L1 (such as proliferating lymphoid tissues) as positive controls. Specific NHP2L1 staining should predominantly localize to the nucleus, with particular enrichment in nucleoli, reflecting its role in splicing and ribosome biogenesis. When optimizing protocols, test multiple antigen retrieval methods; EDTA buffer at pH 9 typically provides optimal results. If non-specific staining persists, increase antibody dilution (1/1500 rather than 1/1000) and consider using a biotin-free detection system to minimize background. Finally, competitive blocking with the immunizing peptide can confirm staining specificity in questionable cases .

How should researchers interpret changes in NHP2L1 expression or localization in disease models?

When interpreting changes in NHP2L1 expression or localization in disease models, researchers should consider several factors. First, as NHP2L1 functions in fundamental cellular processes (splicing and ribosome biogenesis), alterations may reflect broader changes in cellular metabolism rather than disease-specific effects. Changes in subcellular localization—particularly redistribution from nucleoli to nucleoplasm or cytoplasm—may indicate disruption of either spliceosome assembly or SSU processome function. When quantifying expression changes by Western blot or immunohistochemistry, normalize to multiple housekeeping genes/proteins, as traditional references may themselves be affected by altered ribosome biogenesis. Finally, correlate NHP2L1 changes with functional readouts of splicing efficiency (exon inclusion/skipping) and ribosome production (nucleolar integrity, pre-rRNA processing) to establish mechanistic relationships rather than merely associative findings .

What is known about small molecule inhibitors of NHP2L1-RNA interactions?

Research has identified topotecan and other camptothecin derivatives as potent disruptors of the NHP2L1-U4 RNA interaction. These compounds were discovered through a high-throughput screening campaign using time-resolved FRET to detect the interaction between NHP2L1 and the U4 5' stem loop. Mechanistic studies revealed that topotecan binds directly to U4 RNA rather than to NHP2L1 protein, as confirmed through NMR spectroscopy experiments. The disruption of this critical interaction inhibits RNA splicing, revealing a previously uncharacterized mechanism of action for these compounds beyond their well-known topoisomerase inhibition. This finding opens new therapeutic possibilities for modulating splicing in diseases characterized by splicing dysregulation .

How might NHP2L1 contribute to cancer biology and other disease mechanisms?

NHP2L1's fundamental roles in ribosome biogenesis and pre-mRNA splicing position it as a potential contributor to cancer biology and other diseases characterized by dysregulated gene expression. Cancer cells typically exhibit heightened ribosome biogenesis to support their increased protein synthesis demands, suggesting NHP2L1 may be upregulated in rapidly proliferating malignancies. Additionally, alterations in splicing patterns are increasingly recognized as contributors to cancer development and progression. NHP2L1 dysfunction could potentially alter the splicing of oncogenes, tumor suppressors, or genes involved in cell cycle regulation, apoptosis, or DNA repair. While direct evidence linking NHP2L1 to specific disease mechanisms remains limited, its position at the intersection of two fundamental RNA processing pathways warrants further investigation in the context of cancer and other disorders characterized by ribosome biogenesis or splicing aberrations .

What potential exists for developing NHP2L1-targeted therapeutics?

The discovery that topotecan can disrupt the NHP2L1-U4 RNA interaction suggests potential for developing targeted therapeutics aimed at modulating splicing through this mechanism. While direct NHP2L1 inhibition might cause broad cellular toxicity due to its essential functions, targeted disruption of specific NHP2L1 interactions could provide more selective effects. RNA-binding small molecules designed to interfere with the NHP2L1-U4 5' stem loop interaction represent a promising approach. Structure-based drug design informed by crystallographic and NMR data on the NHP2L1-RNA complex could yield compounds with improved specificity and reduced off-target effects compared to topotecan. Additionally, antisense oligonucleotides or RNA-targeting CRISPR systems could potentially modulate specific NHP2L1-dependent splicing events without completely abolishing NHP2L1 function, offering precision approaches for diseases characterized by splicing dysregulation .