NUP96 Antibody

What is NUP96 and why is it significant for molecular research?

NUP96 is a component of the nuclear pore complex (NPC), specifically part of the Nup107-160 complex, which is the largest subunit of the NPC. It is first synthesized as a 186-198 kDa Nup98-Nup96 precursor protein that undergoes rapid autoproteolysis to generate two separate nucleoporins: the amino-terminal Nup98 (Nup98-N) and carboxy-terminal Nup96 (Nup96-C). Both proteins localize to the nucleoplasmic side of the NPC, with NUP96 present in 32 copies per nuclear pore, forming cytoplasmic and nucleoplasmic rings . This well-defined stoichiometry and localization pattern make NUP96 an excellent reference standard for quantitative microscopy and studies of nuclear transport .

What applications are NUP96 antibodies validated for?

NUP96 antibodies have been extensively validated for multiple applications in molecular and cellular biology research. For example, the polyclonal antibody 12329-1-AP has been validated for:

These applications enable researchers to study NUP96 expression, localization, and interactions in various experimental contexts .

How do I choose between polyclonal and monoclonal NUP96 antibodies?

The choice depends on your specific application and research goals:

Polyclonal antibodies like 12329-1-AP recognize multiple epitopes and typically provide stronger signals, making them excellent for applications where sensitivity is crucial. They're particularly useful for detecting proteins in their native state and in immunoprecipitation experiments .

Monoclonal antibodies like MAb 4H5 recognize a single epitope, offering higher specificity and consistency between experiments. The MAb 4H5 specifically recognizes Nup96 with a characteristic nuclear rim staining pattern, making it ideal for immunolocalization studies and analyses requiring high reproducibility .

Consider using monoclonal antibodies when specificity is paramount and polyclonal antibodies when detection sensitivity is the primary concern.

What are the optimal dilution ratios for different applications?

Optimizing antibody dilution is critical for successful experiments. Based on validation data, recommended dilutions for the 12329-1-AP NUP96 antibody are:

It's essential to titrate the antibody in your specific experimental system as optimal concentrations may be sample-dependent .

What antigen retrieval methods are recommended for immunohistochemistry?

For optimal IHC results with NUP96 antibody 12329-1-AP, the recommended antigen retrieval method is:

Primary method: TE buffer at pH 9.0 with heat-mediated antigen retrieval
Alternative method: Citrate buffer at pH 6.0

These protocols have been validated on human breast cancer tissue, demonstrating effective nuclear rim staining patterns characteristic of nuclear pore complex proteins .

How can I optimize immunofluorescence protocols for NUP96 detection?

For optimal immunofluorescence results:

• Use 4% PFA fixation for cultured cells (validated in MCF-7 cells)

• Begin with a 1:200 dilution of primary antibody (12329-1-AP)

• Incubate at room temperature for appropriate time (typically 1-2 hours)

• Use a compatible fluorophore-conjugated secondary antibody (e.g., CoraLite®488-Conjugated AffiniPure Goat Anti-Rabbit IgG for rabbit primary antibodies)

• Look for the characteristic nuclear rim staining pattern

• Compare staining patterns to NPC reference images for validation

Why might I observe different molecular weights for NUP96 in Western blots?

Discrepancies in NUP96 molecular weight observations are common and can be explained by several factors:

• The calculated molecular weight of NUP96 is 98 kDa, but it typically migrates at approximately 105 kDa in SDS-PAGE

• The NUP98-NUP96 precursor is 186-198 kDa before autoproteolysis

• Antibodies targeting different regions may detect different processed forms

• Post-translational modifications can alter migration patterns

• The rapid autoproteolysis process means the full precursor is rarely detected in standard lysates

When troubleshooting, consider which form of the protein your antibody recognizes - antibodies raised against the carboxy-terminal region of Nup98-Nup96 recognize all Nup98-Nup96 proteins except Nup98-N .

How can I distinguish between NUP96 and related proteins like ADAR2?

It's important to note that while Nup98-Nup96 is sometimes known as ADIR2 or ADAR2, it should not be confused with RNA-editing deaminase 1 (Gene ID: 104), which is also named ADAR2. These are distinct proteins with different functions .

To avoid misidentification:

• Verify the specific gene ID (NUP96 is GeneID: 4928, UNIPROT ID: P52948)

• Confirm the expected molecular weight (approximately 105 kDa for NUP96)

• Use validated antibodies with demonstrated specificity

• Look for the characteristic nuclear rim staining pattern in immunofluorescence experiments

• Include appropriate positive controls (e.g., COLO 320 cells, MCF-7 cells)

What controls should I include when investigating protein-protein interactions with NUP96?

When studying NUP96 interactions:

• Input controls: Always include input lysate samples to confirm the presence of target proteins

• Negative controls: Use IgG from the same species as your antibody to identify non-specific binding

• Reciprocal co-IPs: Perform reverse pull-downs using antibodies against suspected interaction partners

• Competitive controls: Pre-incubation with the immunizing peptide can validate specificity

• Interaction validation: Verify interactions using multiple methods (e.g., proximity ligation assays or FRET)

For example, researchers successfully demonstrated interactions between Nup96, HOS1, and HDA6 using Co-IP assays with a 35S:HDA6-MYC transgenic line and an anti-MYC antibody .

How can I use NUP96 as a reference standard for quantitative superresolution microscopy?

NUP96 serves as an excellent calibration tool for superresolution microscopy due to its well-defined stoichiometry and spatial arrangement:

• NUP96 is present in 32 copies per NPC, forming cytoplasmic and nucleoplasmic rings

• Each ring has 8 corners containing two NUP96 molecules positioned 12 nm apart

• This precise architecture provides a built-in molecular ruler at the nanoscale

Researchers have successfully developed cell lines with endogenously tagged NUP96 that allow:

• Quantification of microscope performance and resolution

• Measurement of spatial calibration accuracy

• Determination of absolute labeling efficiencies

• Optimization of imaging conditions

• Precise counting of proteins within complexes

When imaging NUP96 with superresolution techniques, the measured copy numbers should approach the expected values of 16 per ring and 32 per NPC, serving as validation of both the methodology and instrument performance .

How can I accurately quantify NUP96 protein abundance in different genetic backgrounds?

For precise quantification of NUP96 across genetic backgrounds:

• Use standardized sample preparation protocols to ensure consistent extraction

• Include appropriate loading controls for normalization

• Employ quantitative Western blot techniques with standard curves

• Consider fluorescence-based Western blot systems for wider dynamic range

• Use image analysis software to quantify band intensities objectively

• Analyze biological replicates with appropriate statistical tests

• Validate findings with orthogonal methods (e.g., mass spectrometry)

Research has shown that NUP96 abundance can vary significantly across different genetic backgrounds. For example, studies in plants demonstrated that the extent of decrease in Nup96 protein abundance in hos1-3 and nup160-3 mutants was more pronounced than in nup107-3 or nup85-1 mutants, correlating with differential effects on flowering time .

What are the implications of NUP96 tagging strategies for functional studies?

Protein tagging can significantly impact function and experimental outcomes:

• Epitope location matters: C-terminal vs. N-terminal tags can differentially affect protein function

• Tag size considerations: Larger tags may interfere with protein interactions or complex formation

• Validation requirements: Always verify that tagged proteins retain normal localization and function

Evidence from research on related nucleoporins demonstrates this importance. For instance, C-terminal tagging of Nic96 (a NUP96 interaction partner) resulted in measurement of only 26.8 ± 1.2 copies per NPC, contradicting previous reports of 32 copies. When Nic96 was tagged at its N-terminus instead, researchers measured 32.9 ± 2.1 copies, confirming that C-terminal tagging impeded function. This finding was further validated when introducing an additional GFP tag at the C-terminus of Nup49 (which interacts with Nic96's C-terminus) reduced the detectable Nic96 copies to 27.8 ± 1.7 even when Nic96 itself was N-terminally tagged .

These observations underscore the importance of carefully designing tagging strategies and validating their effects on protein function and complex assembly.

How is NUP96 antibody being used to investigate nuclear pore complex assembly?

NUP96 antibodies are valuable tools for studying NPC assembly mechanisms:

• They can track the incorporation of NUP96 into assembling NPCs during cell cycle progression

• Co-staining with other nucleoporin antibodies helps establish assembly hierarchies

• Time-course experiments can reveal the dynamics of precursor processing and integration

• Combined with genetic approaches, they can identify factors required for proper NPC assembly

Researchers have used NUP96 antibodies to demonstrate that it is a component of the Nup107-160 complex, the largest subunit of the nuclear pore complex, providing insight into the architectural organization of NPCs .

What is the significance of NUP96 in gene expression regulation studies?

Recent research has uncovered roles for NUP96 beyond structural components of the NPC:

• NUP96 may form complexes with chromatin-modifying enzymes like HOS1 and HDA6

• Changes in NUP96 protein levels correlate with altered development timing (e.g., flowering time in plants)

• The protein appears to function as a platform for regulatory complexes

For example, Co-IP assays with transgenic plant lines revealed that NUP96 can form complexes with HOS1 and HDA6, suggesting a functional role in gene expression regulation beyond its structural role in nuclear pores .

How can I investigate post-translational modifications of NUP96?

To study NUP96 post-translational modifications:

• Use specialized lysis buffers containing phosphatase and deacetylase inhibitors to preserve modifications

• Employ phospho-specific or other modification-specific antibodies if available

• Consider enrichment techniques like phosphoprotein purification columns

• Analyze by mass spectrometry for comprehensive modification profiling

• Use 2D gel electrophoresis to separate different modified forms

• Compare modification patterns across different cellular conditions (e.g., cell cycle stages, stress responses)

• Validate findings with site-directed mutagenesis of modification sites

While the provided search results don't specifically address NUP96 post-translational modifications, the observed differences between calculated and observed molecular weights (98 kDa vs. 105 kDa) suggest their presence and potential functional significance .