NUP93 Antibody, Biotin conjugated

What is NUP93 and why is it significant in cellular research?

NUP93 (Nucleoporin 93) is a key component of the nuclear pore complex (NPC) that plays critical roles in nuclear transport and cellular function. It forms part of the p62 complex (composed of NUP62 and NUP54) and directly interacts with NUP53, NUP155, NUP205, and lamin B . Recent research has revealed that beyond its structural role in the NPC, NUP93 directly and specifically controls gene transcription by facilitating full BRD4 recruitment to active enhancers . Additionally, NUP93 has been implicated in cancer progression, particularly in aggressive breast cancer subtypes, where its overexpression correlates with poor patient prognosis .

What are the advantages of using biotin-conjugated NUP93 antibodies over unconjugated versions?

Biotin-conjugated NUP93 antibodies offer several methodological advantages:

• Enhanced sensitivity through signal amplification via streptavidin-based detection systems

• Increased flexibility in experimental design (compatible with multiple detection methods)

• Improved stability in various buffers and experimental conditions

• Reduced background in multiplex immunostaining experiments

• Compatibility with proximity-dependent biotinylation (BioID) approaches for studying protein-protein interactions within the NPC context

How do I validate the specificity of a biotin-conjugated NUP93 antibody?

Validation should include:

• Western blot analysis showing a single band at approximately 93 kDa

• Comparison with multiple antibodies targeting different epitopes of NUP93

• Knockdown experiments using siRNA or shRNA against NUP93 (as demonstrated in studies showing reduced viability of cells with NUP93 depletion)

• Immunoprecipitation followed by mass spectrometry to confirm binding to expected interaction partners (NUP53, NUP155, NUP205)

• Immunofluorescence showing characteristic nuclear envelope/nuclear pore staining pattern with colocalization with established NPC markers like mAb414

What are the optimal fixation and permeabilization conditions for immunofluorescence with biotin-conjugated NUP93 antibodies?

For optimal results:

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

• Permeabilize with 0.2-0.5% Triton X-100 for 5-10 minutes

• For improved nuclear envelope visualization, consider a pre-extraction step using 0.5% Triton X-100 in CSK buffer before fixation

• Block with 5-10% normal serum containing 0.1% Triton X-100

• For paraffin-embedded tissues, antigen retrieval using high pressure in citrate buffer (pH 6.0) is recommended

• Dilution ranges from 1:200 to 1:500 for IHC applications, with optimization required for biotin-conjugated formats

How can biotin-conjugated NUP93 antibodies be applied in proximity labeling experiments?

Proximity-dependent biotinylation (BioID) with NUP93:

• Express BirA*-tagged NUP93 in growth-arrested cells (to minimize artifacts from mitotic NPC disassembly)

• Add biotin (50 μM) to culture medium for 18 hours to induce biotinylation

• Harvest cells and isolate biotinylated proteins using streptavidin pull-down

• Analyze by mass spectrometry to identify proximate proteins

• For NUP93, expected interaction partners include components of the Nup93 complex (NUP53, NUP155, NUP205, NUP188) and other nucleoporins as shown in this data table :

What dilutions and blocking conditions are recommended for Western blotting with biotin-conjugated NUP93 antibodies?

Optimal Western blot conditions:

• Recommended dilution range: 0.04-0.4 μg/mL for primary antibody

• Block membranes with 5% non-fat milk or 3-5% BSA in TBST for 1 hour at room temperature

• To minimize background from endogenous biotinylated proteins, pre-block with unconjugated streptavidin

• Include appropriate controls: positive control (cell lysate known to express NUP93), negative control (NUP93-depleted cell lysate)

• For detection, use streptavidin-HRP or streptavidin conjugated to fluorescent dyes

• Expected molecular weight: 93 kDa

How can biotin-conjugated NUP93 antibodies be used to investigate NUP93's role in gene transcription regulation?

Recent research has revealed NUP93's direct role in transcriptional regulation . To investigate this:

• Combine ChIP-seq using biotin-conjugated NUP93 antibodies with Cut&Run techniques to map NUP93 chromatin association

• Compare binding patterns with transcription factors and chromatin modifiers, particularly BRD4

• Use PRO-Seq (precision run-on sequencing) to correlate NUP93 binding with active transcription

• Implement dCas9 chromatin tethering approaches to test causal relationships between NUP93 binding and gene activation

• Analyze NUP93 binding at active promoters and enhancers in different cell types to identify cell-type-specific patterns

• Design experiments to test NUP93's impact on RNA polymerase II loading and transcriptional elongation

What are the considerations for using biotin-conjugated NUP93 antibodies in studying NPC assembly dynamics?

For studying NPC assembly:

• Use biotin-conjugated NUP93 antibodies in conjunction with tracking nanobodies (t-Nbs) that bind soluble Nup complexes and intact NPCs

• Implement rapid degradation systems (e.g., auxin-inducible degrons) to study acute depletion effects

• Design time-course experiments to track NUP93 incorporation during post-mitotic NPC assembly

• Consider the nucleoporin-binding nanobody approach described by researchers that can either track or inhibit nuclear pore complex assembly

• For real-time imaging, combine with complementary fluorescently-labeled NPC components

• Control for potential steric hindrance issues that might affect detection of assembly intermediates

How can discrepancies in NUP93 localization data from different studies be reconciled in experimental design?

Resolving contradictory data:

• The literature shows variance in NUP93 localization data. Some studies locate it exclusively at the nuclear NPC side (35-55 nm from NE midplane), while others show staining patterns on both sides of the NPC

• To address these discrepancies:

  • Use multiple antibodies targeting different epitopes of NUP93

  • Implement higher-resolution techniques like direct gold-coupling of primary antibodies or Fab fragments to reduce signal spread

  • Compare results from different fixation and sample preparation methods

  • Consider that different cell types or physiological states may affect NUP93 localization

  • Account for technical differences between immuno-EM methods that may contribute to these variances

  • Use complementary approaches like proximity-dependent biotinylation to map the spatial organization of NUP93 relative to other NPC components

What are the common causes of high background when using biotin-conjugated NUP93 antibodies, and how can they be minimized?

Common issues and solutions:

• Endogenous biotinylated proteins: Pre-block with unconjugated streptavidin or avidin

• Non-specific binding: Optimize blocking conditions (5% BSA or 10% normal serum from the same species as secondary reagent)

• Signal amplification issues: Titrate streptavidin-detection reagent concentration

• Buffer compatibility problems: Avoid PBS with high biotin content; use TBS when possible

• Storage degradation: Aliquot antibody upon receipt and store at -20°C; avoid repeated freeze-thaw cycles

• For immunohistochemistry applications, include additional blocking steps with avidin-biotin blocking kits

• Consider the buffer composition: Tris-citrate/phosphate buffer, pH 7-8 is used for commercial antibody formulations

How can I optimize co-immunoprecipitation protocols using biotin-conjugated NUP93 antibodies to study nuclear pore complex assembly?

Optimized co-IP approach:

• Use mild lysis conditions (e.g., 0.5-1% NP-40 or 0.5% Triton X-100) to preserve protein-protein interactions

• Include appropriate salt concentration (150-300 mM NaCl) to reduce non-specific binding while maintaining complex integrity

• For studying NUP93 interactions with NUP53 (which is direct), consider crosslinking approaches

• When investigating interactions with other Y-complex components, follow protocols similar to those used for HA-Nup43 and GFP-Y-Nups co-transfection experiments

• Utilize streptavidin-coated magnetic beads for efficient pull-down

• Include RNase treatment to eliminate RNA-mediated interactions

• For detecting transient interactions, consider using chemical crosslinkers before cell lysis

• Include appropriate controls: IgG control, input sample, and when possible, samples with NUP93-depleted cells

What are the considerations for using biotin-conjugated NUP93 antibodies in dual or triple immunofluorescence labeling experiments?

For multiplexing strategies:

• Consider the order of primary antibody application to avoid steric hindrance

• If combining with other biotin-conjugated antibodies, sequential detection using different fluorophore-conjugated streptavidins may be required with intervening blocking steps

• When co-staining with other NPC components, be aware of potential epitope masking due to the dense structure of the NPC

• For co-localization with other NPC markers like mAb414 (which detects FXFG repeat-containing nucleoporins) or anti-Nup153, optimize antibody dilutions to achieve balanced signal intensities

• Include appropriate controls for each detection channel

• Consider spectral imaging and linear unmixing for closely overlapping fluorophores

• For triple labeling, design experiments that include NUP93 along with markers for both the cytoplasmic and nuclear sides of the NPC

How should researchers interpret changes in NUP93 localization or expression in cancer progression studies?

Interpretation guidelines:

• NUP93 overexpression has been shown to enhance transendothelial migration, matrix invasion, tumor growth, and metastasis

• Analyze NUP93 staining patterns in relation to tumor grade and patient outcome data

• Consider the interaction between NUP93 and TGF-β signaling pathway components when interpreting results

• Assess co-localization with other cancer progression markers

• Quantify nuclear envelope versus intranuclear distribution of NUP93 in different tumor stages

• Correlate NUP93 levels with patient survival data for prognostic assessment

• Compare expression in primary tumors versus metastatic sites to understand its role in cancer progression

What are the limitations of proximity labeling approaches using NUP93 as a bait protein, and how can these be addressed in experimental design?

Limitations and solutions:

• The BioID labeling radius has been measured to be approximately 10-15 nm, which sets spatial constraints on protein detection

• False negatives may occur for transient interactions or spatially distant components

• The dense structure of the NPC may limit accessibility of the biotin ligase to potential interactors

• To address these limitations:

  • Use multiple BioID fusion constructs targeting different domains of NUP93

  • Compare results with complementary approaches like conventional IP-MS

  • Consider split-BioID approaches for detecting specific interaction interfaces

  • Use the Nup107-160 complex as a molecular ruler for calibrating labeling distances as demonstrated in previous studies

  • Account for cell cycle effects by conducting experiments in growth-arrested cells

How can researchers distinguish between NUP93's structural role in the NPC and its gene regulatory functions when analyzing experimental data?

Analytical approaches:

• Implement domain-specific mutations that selectively disrupt either NPC incorporation or chromatin binding

• Use ChIP-seq and RNA-seq to correlate NUP93 chromatin binding with transcriptional changes

• Apply auxin-inducible rapid degradation systems to study acute depletion effects, separating immediate impacts (likely direct) from long-term consequences

• Compare NUP93 chromatin association maps with other NPC components to identify unique patterns

• Analyze cell cycle-specific effects, as NPC disassembly during mitosis provides a natural separation of functions

• Design rescue experiments with domain-specific mutants to attribute phenotypes to specific functions

• Consider the temporal dynamics of NUP93's dual roles, as shown in acute depletion studies where transcriptional effects were observed prior to structural consequences

How might biotin-conjugated NUP93 antibodies contribute to understanding the role of NPCs in neurodegenerative diseases?

Emerging research avenues:

• NPCs show remarkable longevity in post-mitotic neurons, making them potential sites for age-related damage accumulation

• Biotin-conjugated NUP93 antibodies can help track NPC integrity in aging neural tissues

• Design experiments comparing NUP93 distribution and post-translational modifications in healthy versus diseased brain tissues

• Investigate potential correlations between NUP93 alterations and nucleocytoplasmic transport defects observed in neurodegenerative conditions

• Develop protocols for multiplexed detection of NUP93 and disease-specific protein aggregates

• Implement super-resolution microscopy approaches to detect subtle changes in NPC architecture during disease progression

What methodological advances are needed to better understand the dynamic interactions between NUP93 and chromatin during gene regulation?

Future methodological needs:

• Development of live-cell imaging approaches using split-fluorescent protein systems fused to NUP93 and chromatin components

• Implementation of single-molecule tracking to monitor NUP93 dynamics at specific genomic loci

• Advancement of high-resolution chromosome conformation capture methods (Hi-C, HiChIP) to map 3D genome contacts mediated by NUP93

• Creation of optogenetic tools to temporally control NUP93 function in specific cellular compartments

• Design of CRISPR-based approaches to visualize endogenous NUP93 interactions with specific genomic regions

• Development of computational models integrating multiple datasets to predict NUP93's gene regulatory impact

How can biotin-conjugated NUP93 antibodies be integrated with mass spectrometry approaches to map post-translational modifications affecting NUP93 function?

Integrated approaches:

• Combine immunoprecipitation using biotin-conjugated NUP93 antibodies with mass spectrometry to identify PTMs

• Implement targeted mass spectrometry approaches (MRM/PRM) to quantify specific modifications under different cellular conditions

• Compare PTM profiles between normal and disease states to identify functionally relevant modifications

• Develop modification-specific antibodies based on MS findings to track specific NUP93 subpopulations

• Integrate proteomic data with functional assays to determine how specific modifications affect NUP93's dual roles in NPC structure and gene regulation

• Consider crosslinking mass spectrometry (XL-MS) approaches to map interaction interfaces as mentioned in NPC organization studies