NUP50B Antibody

What is NUP50 and what is its function in the cell?

NUP50 (also known as Npap60) is a mobile nucleoporin that localizes primarily in the nuclear basket of the nuclear pore complex (NPC) and in the nucleoplasm. It functions by binding directly to importin α, and this interaction plays a crucial role in nuclear import processes. NUP50 is positioned on the nucleoplasmic side of the NPC, where it participates in the regulation of macromolecular transport between the nucleus and cytoplasm .

Human NUP50 exists in two isoforms: Npap60L (1-469 amino acids) and its alternatively spliced isoform, Npap60S (29-469 amino acids) . Electron microscopy studies have shown that NUP50 is located at or near the nucleoplasmic fibrils of the NPC, in proximity to where Nup153 has been localized, with most gold particle labeling occurring between 15 and 80 nm from the midplane of the NPC .

What applications are NUP50 antibodies typically used for?

NUP50 antibodies are primarily used in several key research applications:

The antibody has been positively tested in Western blotting for multiple cell lines including HCT 116, HeLa, HEK-293, Ramos, Daudi, Sp2/0, MCF-7, HT-29, U2OS, HepG2, Jurkat, MOLT-4, and K-562 cells . For immunohistochemistry, it's recommended to use TE buffer pH 9.0 for antigen retrieval, though citrate buffer pH 6.0 can also be effective .

What is the molecular weight of NUP50 and how is it observed in experimental conditions?

NUP50 has a calculated molecular weight of 50 kDa (468 amino acids), but is typically observed at 50-55 kDa in experimental conditions such as Western blotting . This slight discrepancy between calculated and observed molecular weights is common for many proteins and can be attributed to post-translational modifications or the inherent properties of the protein that affect its migration in SDS-PAGE gels.

How can I optimize Western blot protocols for NUP50 detection?

For optimal Western blot detection of NUP50, follow these methodological considerations:

• Sample preparation: Use cell lysates from cell lines known to express NUP50, such as HeLa, HEK-293, or other validated cell lines listed in the antibody specifications.

• Antibody dilution: Begin with a mid-range dilution (1:10000-1:20000) and adjust based on signal intensity. The recommended range is 1:5000-1:50000 .

• Blocking: Use a standard blocking buffer with either 5% non-fat dry milk or BSA in TBST.

• Visualization: Both chemiluminescence and fluorescence-based detection systems are suitable depending on your laboratory's equipment.

• Controls: Include positive controls from validated cell lines and consider using recombinant NUP50 as a standard if absolute quantification is needed.

Remember that optimal conditions may need to be titrated for each specific experimental system to obtain the best results .

What methods can be used to study NUP50 localization at the subcellular level?

Several complementary approaches can be employed to study NUP50 localization:

• Immunofluorescence microscopy: Using anti-NUP50 antibodies on fixed and permeabilized cells. Note that permeabilization method matters - Triton X-100 is needed to access the nucleoplasmic side of the NPC where NUP50 is located, as digitonin permeabilization (which only permeabilizes the plasma membrane) is insufficient .

• Immunogold electron microscopy: This provides high-resolution localization data. Pre-embedding immunogold labeling with anti-NUP50 antibodies on isolated nuclear envelopes or permeabilized cells allows precise localization of NUP50 to the nucleoplasmic side of the NPC .

• Cell fractionation: Biochemical isolation of nuclear envelope fractions followed by Western blotting can confirm the presence of NUP50 in nuclear compartments.

• Live-cell imaging: Using fluorescently tagged NUP50 constructs to track its dynamics in living cells.

Research has shown through immunogold electron microscopy that NUP50 localizes almost exclusively to the nucleoplasmic surface of the NPC, with labeling patterns that resemble rings in tangential views, similar to antigens localized in the terminal ring structure of the nucleoplasmic basket of the NPC .

How can I perform immunoprecipitation with NUP50 antibodies to identify interaction partners?

To identify proteins that interact with NUP50, follow this immunoprecipitation protocol based on published methods:

• Cell preparation: Permeabilize approximately 4 × 10^6 cells (e.g., NRK cells) with digitonin.

• Cell lysis: Solubilize cells in buffer containing 1% NP-40, 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl₂, 60 mM β-glycerophosphate, 2 mM DTT, and protease inhibitors (1 μg/ml each of leupeptin, pepstatin, and aprotinin).

• Clarification: Centrifuge at 100,000 × g for 30 minutes.

• Antibody addition: Add purified anti-NUP50 antibody to the solubilized cell supernatant at 5-10 μg/ml and incubate for 2 hours at 4°C.

• Immunoprecipitation: Collect antibody-antigen complexes using protein A-Sepharose.

• Analysis: Analyze immunoprecipitated proteins by SDS-PAGE and immunoblotting with antibodies against suspected interaction partners .

This approach has successfully identified interactions between NUP50 and nuclear transport factors such as CRM1, suggesting a role for NUP50 in nuclear export pathways .

What are common technical challenges when working with NUP50 antibodies and how can they be addressed?

When working with NUP50 antibodies, researchers may encounter several technical challenges:

• Cross-reactivity issues: Some anti-NUP50 antibodies may recognize related proteins. For instance, one batch of anti-NUP50 antibodies (referred to as antibody 1 in the literature) recognized both NUP50 and a 70 kDa protein (p70) . To address this:

  • Always validate antibody specificity by Western blotting

  • Consider using antibodies raised against different epitopes of NUP50

  • Use recombinant NUP50 as a positive control

• Accessibility in fixed specimens: NUP50 is located on the nucleoplasmic side of the NPC, making it inaccessible to antibodies in cells permeabilized only with digitonin. For successful immunolabeling:

  • Use Triton X-100 for permeabilization when performing immunofluorescence

  • For electron microscopy, use freeze-thawing or other methods that ensure access to nucleoplasmic antigens

• Optimal fixation methods: Different fixation protocols may affect epitope accessibility:

  • Test both paraformaldehyde and methanol fixation

  • For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can also be used

How do I distinguish between specific and non-specific signals when using NUP50 antibodies?

To distinguish between specific and non-specific signals:

• Use appropriate controls:

  • Include a blocking peptide control where excess antigen is pre-incubated with the antibody before application

  • Include samples known to be negative for NUP50 expression

  • Use secondary-only controls to assess background from secondary antibodies

• Validate with multiple detection methods:

  • Confirm Western blot results with immunofluorescence or vice versa

  • When possible, use genetic approaches (knockdown/knockout) to validate specificity

• Competition assays:

  • As demonstrated in microinjection experiments, adding a mixture of antibody and excess antigen used for immunization can restore normal function, confirming antibody specificity

• Look for expected localization and molecular weight:

  • Specific signal should show nuclear envelope/nuclear pore staining

  • In Western blots, bands should appear at 50-55 kDa

How does NUP50 contribute to nuclear export mechanisms and what methods can be used to study this function?

NUP50 plays a significant role in nuclear export pathways, particularly those mediated by CRM1 (Exportin-1). Research has revealed:

• Direct involvement in export: Microinjection of anti-NUP50 antibodies into cell nuclei strongly inhibits the export of proteins containing leucine-rich nuclear export signals (NES) .

• Specificity for CRM1-mediated export: A recombinant fragment of NUP50 containing several FG repeat motifs selectively binds to CRM1 (but not to importin β or the export receptor CAS) in the presence of NES cargo and RanGTP .

To study NUP50's role in nuclear export, researchers can employ:

• Microinjection assays: Inject anti-NUP50 antibodies along with fluorescently labeled export substrates into cell nuclei and monitor substrate distribution over time. In control cells, NES-containing substrates become concentrated in the cytoplasm, while in cells injected with anti-NUP50 antibodies, substrates remain confined to the nucleus .

• In vitro binding assays: Couple recombinant NUP50 fragments to Sepharose beads and test binding of purified transport receptors (CRM1, importin β, CAS) in the presence or absence of cargo and RanGTP. Analyze bound proteins by SDS-PAGE and immunoblotting .

• CRISPR-mediated knockout/knockdown: Modern approaches using gene editing to deplete NUP50 can reveal its role in various transport pathways.

• Super-resolution microscopy: Track the dynamics of export complexes relative to NUP50 location at the NPC.

What is known about the structural domains of NUP50 and how do they contribute to its function?

NUP50 contains several functional domains that contribute to its role in nucleocytoplasmic transport:

• FG repeat motifs: These phenylalanine-glycine repeats are characteristic of many nucleoporins and serve as docking sites for transport receptors. In NUP50, these repeats are important for binding to CRM1 during nuclear export .

• Ran-binding domain: NUP50 can interact with the small GTPase Ran, which regulates the directionality of nuclear transport.

• Importin α-binding region: NUP50 binds directly to importin α, playing a role in nuclear import pathways.

Human NUP50 exists in two isoforms due to alternative splicing:

• Npap60L (1-469 amino acids): The longer, full-length isoform

• Npap60S (29-469 amino acids): A shorter isoform missing the first 28 amino acids of Npap60L

Functional studies using recombinant fragments have shown that a region containing amino acids 173-357 is sufficient for some interactions, including being recognized by antibodies . Structure-function studies combining domain deletion/mutation with functional assays would be valuable for further elucidating the specific contributions of each domain to NUP50's role in nuclear transport.

How can mass spectrometry be used to identify NUP50-associated proteins in different cellular contexts?

Mass spectrometry is a powerful approach for identifying NUP50-associated proteins. Based on methodologies described in the literature, researchers can follow this workflow:

• Immunoprecipitation of NUP50 complexes:

  • Solubilize cells in appropriate lysis buffer

  • Immunoprecipitate using anti-NUP50 antibodies

  • Separate proteins by SDS-PAGE

• Sample preparation for mass spectrometry:

  • Excise gel bands of interest

  • Perform in-gel trypsin digestion

    • Immerse gel strips in 25 mM ammonium bicarbonate/50% acetonitrile

    • Shake for 10 minutes, remove solution, and rinse

    • Dry completely under nitrogen

    • Rehydrate and add 0.5 mg modified sequence grade trypsin

    • Incubate overnight with agitation at 30°C

  • Transfer supernatants to new tubes for analysis

• Mass spectrometry analysis:

  • Use MALDI (matrix-assisted laser desorption ionization) technique with α-cyano-4-hydroxy-cinnamic acid matrix

  • Employ time-of-flight analyzer

  • Consider that without a reflectron, an error of 0.1% is possible

  • Mass peaks above 3,000 mass units may need to be excluded from alignment

• Data analysis:

  • Compare detected peptide masses with databases to identify proteins

  • Validate findings with alternative methods (Western blotting, immunofluorescence)

This approach has successfully identified NUP50-associated proteins in previous studies and can be adapted to study NUP50 interactions in different cellular contexts or in response to various stimuli .

What are the optimal storage conditions for NUP50 antibodies?

For maximum stability and activity retention of NUP50 antibodies, follow these storage guidelines:

• Temperature: Store at -20°C. Commercial NUP50 antibodies are typically stable for one year after shipment when stored properly .

• Buffer composition: Most NUP50 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting: While the product information indicates that aliquoting is unnecessary for -20°C storage, it may still be beneficial to prepare small aliquots to avoid repeated freeze-thaw cycles if the antibody will be used multiple times over an extended period .

• Special considerations: Some preparations of NUP50 antibodies (in 20μl sizes) contain 0.1% BSA as a stabilizer .

• Working dilutions: Diluted working stocks should be prepared fresh and used within 24 hours.

Following these guidelines will help ensure consistent results across experiments and maximize the useful lifetime of the antibody.