NUP50A Antibody

What is NUP50 and why are antibodies against it valuable for cellular research?

NUP50 (Nucleoporin 50 kDa, also known as NPAP60) is a mobile nucleoporin primarily localized to the nucleoplasmic side of the nuclear pore complex (NPC) and in the nucleoplasm. This 468 amino acid protein contains multiple phenylalanine-glycine (FG) repeats and a RanBD1 domain . NUP50 functions as a binding site for export receptor-cargo complexes and plays a direct role in CRM1-mediated nuclear protein export .

Antibodies against NUP50 are valuable because they enable researchers to:

• Visualize the three-dimensional localization of NUP50 within the nuclear pore complex

• Study nucleocytoplasmic transport mechanisms

• Investigate NPC composition and dynamics during cellular processes

• Examine potential relationships between NUP50 and disease states

Importantly, NUP50 antibodies have revealed that this protein is not solely associated with the nuclear envelope but can also be found in the nuclear interior, suggesting additional functions beyond its role in the NPC .

How is NUP50 distributed across different cell types and tissues?

NUP50 demonstrates variable expression patterns across tissues and cell types:

While NUP50 was originally thought to be restricted to specific tissues, more sensitive detection methods have revealed that it is widely distributed across cell types, though expression levels vary significantly . In some tissues like kidney and heart, a ~70kDa immunoreactive protein (p70) is detected alongside or instead of the canonical 50kDa NUP50, suggesting the existence of NUP50-related proteins or isoforms .

What are the optimal protocols for immunolocalization of NUP50 using antibodies?

The subcellular localization of NUP50 is highly dependent on fixation techniques, requiring careful methodological consideration:

Methanol-Acetone Fixation:

• Yields exclusively nuclear envelope staining

• May extract or mask epitopes of the nuclear pool of NUP50

• Best for studies focusing on NPC-associated NUP50

• Protocol: Fix cells in ice-cold methanol for 5 minutes followed by acetone for 2 minutes

Paraformaldehyde Fixation:

• Results in bright homogeneous nuclear staining (excluding nucleolar regions)

• Preserves the nuclear pool of NUP50 not associated with the nuclear envelope

• Recommended for studies examining both NPC and nucleoplasmic functions

• Protocol: Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

For comprehensive analysis, researchers should perform both fixation methods in parallel to fully characterize NUP50 distribution patterns in their experimental system.

How can researchers optimize immunogold electron microscopy for NUP50 localization?

Pre-embedding immunogold labeling offers high-resolution localization of NUP50 at the ultrastructural level:

• Sample Preparation:

  • For isolated nuclear envelopes: Suspend NE (~200 A260 U/ml) in PBS containing 0.1% BSA

  • For cells: Subject cell pellets to a single freeze-thaw cycle in liquid nitrogen to permeabilize

• Primary Antibody Incubation:

  • Use affinity-purified anti-NUP50 antibodies (5 μg/ml)

  • Incubate for 2 hours at room temperature

  • For co-localization studies, combine with antibodies against other nucleoporins (e.g., anti-Nup153)

• Secondary Antibody Labeling:

  • After washing, incubate with gold-conjugated secondary antibodies

  • For single labeling: Use goat anti-rabbit IgG with 5-nm gold particles

  • For double labeling: Use both 5-nm and 10-nm gold particles conjugated to different secondary antibodies

  • Incubate for 2-3 hours at room temperature

• Post-Labeling Processing:

  • Wash thoroughly, fix in glutaraldehyde followed by osmium tetroxide

  • Embed in Epon for sectioning

  • Image using transmission electron microscopy at 70-80 kV

This approach has revealed that NUP50 is predominantly localized to the nucleoplasmic side of the NPC, approximately 35-45 nm from the midplane, consistent with localization to the nucleoplasmic fibrils .

What are the critical considerations when selecting between different NUP50 antibody formats?

When selecting NUP50 antibodies, researchers should consider:

Validation experiments should include:

• Western blotting to confirm molecular weight (50-55 kDa)

• Testing in multiple cell lines with known NUP50 expression

• Including knockout or knockdown controls when available

How can NUP50 antibodies be used to investigate nuclear transport mechanisms?

NUP50 antibodies have been instrumental in characterizing nuclear transport pathways:

Microinjection Experiments:

• Prepare affinity-purified anti-NUP50 antibodies in microinjection buffer (10 mM sodium phosphate, pH 7.2, 80 mM KCl, 5% glycerol)

• Co-inject antibodies with fluorescently labeled transport substrates (e.g., GST-NES for export studies)

• Include control injections with buffer alone and with antibody pre-incubated with antigen

• Monitor substrate localization over time (e.g., 30-minute timepoints)

• Quantify nuclear-cytoplasmic distribution using fluorescence microscopy

This approach has revealed that anti-NUP50 antibodies strongly inhibit CRM1-mediated nuclear export but do not affect importin α/β-dependent nuclear import, suggesting a selective role for NUP50 in export pathways .

Biochemical Interaction Studies:

• Couple recombinant NUP50 fragments to CNBr-activated Sepharose beads

• Incubate with purified nuclear transport receptors (e.g., CRM1, importin-β)

• Test binding in the presence or absence of cargo molecules and Ran-GTP

• Analyze bound fractions by SDS-PAGE and immunoblotting

• Compare results with known nucleoporin-transport receptor interactions

These experiments have demonstrated that NUP50 specifically interacts with the export receptor CRM1 in the presence of cargo and Ran-GTP, providing mechanistic insight into its role in nuclear export .

What is the significance of NUP50 in neurodegenerative disease research, particularly ALS?

Recent evidence has implicated NUP50 in amyotrophic lateral sclerosis (ALS) pathogenesis:

Key Findings:

• Loss of nuclear NUP50 immunoreactivity has been observed in ALS patient samples

• Rare NUP50 mutations have been identified in ALS patients

• Nuclear NUP50 is decreased in both SOD1 and FUS animal models of ALS

• Changes in NUP50 may represent a common pathway in different forms of ALS

Research Approaches:

• Immunohistochemical Analysis:

  • Compare NUP50 localization in ALS patient samples versus controls

  • Use semi-quantitative analysis of immunoreactivity to measure NUP50 expression levels

  • Co-stain with TDP-43 antibodies to examine correlation with TDP-43 pathology

• Genetic Studies:

  • Screen for NUP50 mutations in familial and sporadic ALS cases

  • Characterize the functional consequences of identified mutations

  • Develop animal models with NUP50 alterations to assess motor neuron degeneration

• Mechanistic Investigations:

  • Examine the relationship between NUP50 loss and nucleocytoplasmic transport defects

  • Investigate alterations in mRNA export and protein import in affected neurons

  • Test whether restoring NUP50 function can ameliorate disease phenotypes

This emerging research suggests that targeting nucleocytoplasmic transport, including NUP50-dependent pathways, may represent a therapeutic strategy for ALS and related neurodegenerative diseases .

How do developmental studies utilize NUP50 antibodies to understand its essential functions?

NUP50 knockout mouse models have revealed critical developmental roles:

Embryonic Development:

• NUP50 is widely expressed during mouse embryogenesis

• Homozygous NUP50 deletion results in late embryonic lethality

• NUP50-null embryos exhibit neural tube defects and intrauterine growth retardation

• The developing neural tube shows particularly high NUP50 expression

Research Approaches Using NUP50 Antibodies:

• Developmental Expression Analysis:

  • Perform immunohistochemistry on staged embryo sections

  • Quantify NUP50 expression levels in different tissues during development

  • Compare with other nucleoporins to identify unique developmental patterns

• Cell Proliferation Studies:

  • Use BrdU incorporation coupled with NUP50 immunostaining

  • Examine correlation between NUP50 expression and proliferative zones

  • Analyze NUP50-null tissues for proliferation defects

• Cell Cycle Regulator Analysis:

  • Investigate the relationship between NUP50 and cell cycle regulators like p27Kip1

  • Perform co-immunoprecipitation experiments to identify developmental stage-specific interactions

  • Compare wild-type and NUP50-null embryonic fibroblasts for cell cycle parameter differences

These studies have established that while NUP50 is dispensable for basic cellular processes in many cell types, it has essential functions in specific developmental contexts, particularly in the developing nervous system .

How can researchers address common issues with NUP50 antibody specificity and cross-reactivity?

NUP50 antibodies may detect related proteins or show variable specificity. Here are approaches to overcome these challenges:

Problem: Detection of multiple bands (e.g., p70) alongside NUP50

Solution:

• Test multiple antibodies raised against different epitopes of NUP50

• Compare polyclonal (antibody 1, which detects both NUP50 and p70) with more specific antibodies (antibody 2, which detects only NUP50)

• Perform mass spectrometry on immunoprecipitated proteins to confirm identity

• Include knockout or knockdown controls when available

Problem: Variable staining patterns across fixation methods

Solution:

• Use both methanol-acetone and paraformaldehyde fixation in parallel

• Consider that different fixation methods may reveal different pools of NUP50

• Validate localization patterns with multiple antibodies

• Perform subcellular fractionation followed by Western blotting to confirm distribution

Problem: Non-specific background in immunohistochemistry

Solution:

• Optimize antibody concentration (test dilutions from 1:500 to 1:10,000)

• Use appropriate antigen retrieval (TE buffer pH 9.0 recommended for most applications)

• Include blocking steps with BSA or serum from the species of the secondary antibody

• Consider alternate detection systems if background persists

What are the optimal storage and handling conditions for maintaining NUP50 antibody activity?

To maintain optimal antibody performance:

Additional handling considerations:

• Avoid repeated freeze-thaw cycles which can denature antibodies

• For long-term storage of working dilutions, add carrier protein (0.1% BSA)

• Centrifuge briefly before opening to collect solution at the bottom of the vial

• Store conjugated antibodies (FITC, PE) protected from light to prevent photobleaching

How are technological innovations expanding NUP50 antibody applications in research and therapeutics?

The NUP50 antibody field is evolving rapidly with several key technological advances:

High-Affinity Monoclonal Antibodies:

• Enhanced specificity and sensitivity for detecting NUP50

• Improved visualization of NUP50 localization in tissue samples

• Better understanding of functional dynamics in cellular processes

CRISPR Technology Integration:

• Precise gene editing to create knockout models

• Investigation of physiological roles of NUP50 in vivo

• Discovery of novel therapeutic targets

• Deeper understanding of NUP50's involvement in pathophysiological mechanisms

Nanotechnology Delivery Systems:

• Nanoparticles designed to encapsulate NUP50 antibodies

• Improved bioavailability and target specificity

• Reduced off-target effects and enhanced therapeutic efficacy

• Better tumor penetration and reduced systemic toxicity in cancer applications

Multiplex Assays:

• Simultaneous detection of multiple biomarkers including NUP50

• Comprehensive understanding of complex biological systems

• Enhanced biomarker discovery for early disease diagnosis

• Broader analysis of cellular pathways

Computational Biology and Machine Learning:

• Prediction of NUP50 interactions with other proteins

• Analysis of large datasets to uncover patterns and correlations

• Streamlined experimental designs and hypothesis generation

• More efficient research pathways focusing on promising avenues

These technological innovations are collectively advancing both fundamental research into NUP50 biology and the development of potential therapeutic strategies targeting NUP50-related pathways.