IPO11 Antibody

What is IPO11 and what is its primary function in cellular systems?

IPO11 (Importin-11, also known as RanBP11) is a nuclear transport receptor belonging to the karyopherin/importin-β family. It functions primarily in nuclear protein import by:

• Acting as a receptor for nuclear localization signals (NLS) in cargo substrates

• Mediating docking of the importin/substrate complex to the nuclear pore complex (NPC) via nucleoporin binding

• Facilitating translocation through the nuclear pore by an energy-requiring, Ran-dependent mechanism

The directional movement is regulated by an asymmetric distribution of GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus. At the nucleoplasmic side of the NPC, Ran binds to IPO11, causing the importin/substrate complex to dissociate. IPO11 is then re-exported to the cytoplasm where GTP hydrolysis releases Ran .

What are the most effective applications for IPO11 antibodies in research?

IPO11 antibodies have been validated for multiple research applications with varying effectiveness:

For optimal results, antibody selection should be based on the specific application and experimental conditions required .

How should researchers validate IPO11 antibody specificity?

Proper validation of IPO11 antibody specificity should follow these methodological steps:

• Genetic controls: Compare signals between wild-type and IPO11 knockout or knockdown samples. Research has confirmed absence of IPO11 signal in homozygous mutant mice using RT-PCR and western blotting .

• Western blot analysis: Verify a single band at the expected molecular weight (113 kDa). Heterozygous samples should show approximately half the signal intensity of wild-type samples .

• Loading controls: Always include appropriate loading controls (e.g., GAPDH, β-actin) for quantitative comparisons .

• Cross-reactivity testing: If working with multiple species, confirm reactivity with the target species. Most IPO11 antibodies work with human samples, with varying cross-reactivity to mouse, rat, and other species .

• Immunogen verification: Check whether the antibody was raised against a region relevant to your research question. Available antibodies target different regions of IPO11, including N-terminal (AA 1-50), mid-region (AA 328-377), and C-terminal domains (AA 776-975, AA 891-920) .

How does IPO11 contribute to embryonic development, and what methodologies best demonstrate this relationship?

IPO11 plays a critical role in embryonic development, as demonstrated by gene knockout studies:

• Embryonic lethality: Homozygous IPO11 knockout (IPO11-/-) mice die by embryonic day 11.5, demonstrating its essential role in development .

• Experimental approach: The relationship between IPO11 and embryonic development was established using gene trap vector insertion at the 2nd intron of the IPO11 gene, followed by PCR genotyping with specific primer pairs (wild-type allele: 514 bp band; mutant allele: 322 bp band) .

• Expression analysis: RT-PCR analysis using primers for exons 22-30 (forward: 5'-CACACCAGAGCTGCTTCGTA-3', reverse: 5'-TTTCCATGAGGGACTGGAAG-3') with β-actin as internal control can verify expression levels .

• Protein verification: Western blot analysis confirms the absence of IPO11 protein in homozygous embryos, with heterozygotes showing approximately half the protein level compared to wild-type embryos .

For developmental studies, temporal expression profiling using these methods can identify critical windows when IPO11 function becomes essential for embryonic viability.

What is the role of IPO11 in cancer biology, particularly in acute myeloid leukemia (AML)?

IPO11 has emerging significance in cancer biology, with particularly important functions in AML:

• Leukemic stem cell (LSC) maintenance: IPO11 was identified through a CRISPR screen comparing genes significant for growth and viability of AML cells with genes differentially expressed in LSCs .

• Functional consequences of inhibition: Knockdown of IPO11 in AML results in:

  • Decreased cell growth

  • Reduced engraftment potential of leukemic stem cells

  • Induction of differentiation

• Molecular mechanism: IPO11 regulates the nuclear import of transcription factors BZW1 and BZW2, which mediate a transcriptional signature promoting stemness and survival of LSCs .

• Therapeutic implications: Targeting the IPO11-BZW1/2 axis represents a potential novel therapeutic strategy for AML, particularly for addressing the treatment-resistant LSC population associated with relapse .

Research methods for studying IPO11 in cancer include knockdown experiments, transcriptional profiling, protein localization studies, and in vivo models to assess stemness and differentiation.

What are the known cargo proteins of IPO11 and how can researchers identify novel cargo?

IPO11 transports specific cargo proteins from the cytoplasm to the nucleus:

Confirmed cargo proteins:

• UBE2E3 (ubiquitin-conjugating enzyme E2 E3)

• RPL12 (ribosomal protein L12)

• BZW1 and BZW2 (transcription factors involved in AML)

Methods for identifying novel IPO11 cargo proteins:

• Co-immunoprecipitation: Using anti-IPO11 antibodies to pull down complexes, followed by mass spectrometry to identify interacting proteins .

• Proximity labeling: BioID or APEX2 fused to IPO11 to identify proteins in close proximity.

• Nuclear import assays: Monitoring nuclear accumulation of candidate proteins in the presence and absence of IPO11.

• Subcellular fractionation: Comparing nuclear/cytoplasmic distribution of proteins upon IPO11 knockdown or overexpression.

• NLS prediction and validation: Computational prediction of nuclear localization signals in candidate proteins, followed by mutagenesis and localization studies.

When identifying new cargo, researchers should confirm specificity by demonstrating:

• Direct interaction with IPO11

• IPO11-dependent nuclear localization

• Presence of a functional NLS recognized by IPO11

What are the technical considerations for optimizing IPO11 antibody performance in various experimental systems?

Optimizing IPO11 antibody performance requires attention to several technical factors:

For Western Blotting:

• Recommended dilution: 0.5-2 μg/mL or 1:500-1:2000

• Expected molecular weight: 113 kDa

• Sample preparation: Cell lines successfully used include RT-4, U-251 MG, HeLa, 293T, and A-549

• Controls: Mouse tissues (heart, kidney, lung, spleen, testis) serve as good positive controls

For Immunohistochemistry:

• Fixation: Paraformaldehyde (PFA) fixation works well for immunofluorescence

• Permeabilization: Triton X-100 is effective for nuclear protein access

• Recommended dilution: 1:2500 for paraffin-embedded tissues

• Antigen retrieval: May be necessary for formalin-fixed tissues

For Immunoprecipitation:

• Antibody selection: Choose immunoaffinity-purified antibodies

• Bead selection: Protein A/G beads work well with rabbit polyclonal antibodies

• Washing conditions: Optimize to maintain specific interactions while reducing background

General considerations:

• Storage: Most antibodies should be stored at -20°C

• Buffer composition: Typically PBS with 50% glycerol and 0.02% sodium azide at pH 7.3

• Antibody concentration: Typically around 0.29 mg/mL

• Shelf life: Recommended shelf life is generally 1 year from date of receipt

How can researchers effectively use IPO11 antibodies to study the Ran-GTP cycle in nuclear transport?

Studying the Ran-GTP cycle and its regulation of IPO11 function requires specialized experimental approaches:

• Co-immunolocalization studies: Use IPO11 antibodies (4 μg/ml) alongside Ran-GTP specific antibodies to visualize the spatial relationship between IPO11 and Ran-GTP gradients across the nuclear envelope .

• Binding assays: Employ pull-down experiments with IPO11 antibodies under varying Ran-GTP conditions to demonstrate how Ran-GTP affects cargo binding.

• Nuclear transport kinetics: Utilize IPO11 antibodies in permeabilized cell assays where the Ran-GTP gradient can be manipulated to assess transport efficiency.

• Fluorescence recovery after photobleaching (FRAP): Combine with immunofluorescence to study how the Ran-GTP cycle affects IPO11 shuttling dynamics.

• Mutational analysis: Compare wild-type IPO11 with mutants defective in Ran-GTP binding using antibodies that recognize distinct IPO11 epitopes.

For these experiments, researchers should consider using multiple antibodies targeting different IPO11 domains (N-terminal: AA 1-50; C-terminal: AA 776-975) to ensure complete functional characterization .

What are common issues when working with IPO11 antibodies and how can they be resolved?

When troubleshooting, always include proper positive controls where IPO11 is known to be expressed (e.g., U-2 OS, HeLa, and 293T cells) and negative controls (secondary antibody only) .

How can researchers investigate IPO11's role in specific cellular contexts using available antibodies?

To investigate IPO11's role in specific cellular contexts:

• Comparative expression analysis:

  • Use Western blotting (1:500-1:2000 dilution) to quantify IPO11 levels across different cell types, tissues, or disease states

  • Compare with known IPO11-dependent cargo proteins (UBE2E3, RPL12, BZW1/2) to establish functional correlations

• Subcellular localization studies:

  • Employ immunofluorescence (4 μg/ml) to visualize IPO11 distribution and potential colocalization with cargo proteins or nuclear pore components

  • PFA-fixed, Triton X-100 permeabilized cells provide optimal conditions for nuclear transport factor visualization

• Functional perturbation experiments:

  • Combine IPO11 antibody detection with knockdown/knockout approaches to correlate protein levels with phenotypic changes

  • In knockout models, verify complete absence of IPO11 using antibodies against multiple epitopes

• Disease-specific applications:

  • For cancer research, particularly AML, use IPO11 antibodies to assess correlation between expression levels and stemness markers

  • In developmental studies, monitor temporal expression patterns in relation to critical developmental windows

• Interaction network mapping:

  • Use co-immunoprecipitation with IPO11 antibodies followed by mass spectrometry to identify context-specific interaction partners

  • Verify interactions through reciprocal immunoprecipitation and co-localization studies

Remember to select antibodies whose immunogens (e.g., AA 1-50, AA 776-975) are relevant to the specific protein domains or interactions being studied .

What emerging technologies might enhance IPO11 antibody applications in research?

Several emerging technologies show promise for enhancing IPO11 antibody applications:

• Super-resolution microscopy: Techniques like STORM, PALM, and STED can reveal IPO11's precise localization relative to nuclear pore complexes and cargo, overcoming the diffraction limit of conventional microscopy.

• Live-cell imaging with nanobodies: Developing fluorescently tagged nanobodies against IPO11 could enable real-time tracking of nuclear transport dynamics without disrupting function.

• Mass cytometry (CyTOF): Metal-conjugated IPO11 antibodies could allow simultaneous detection of IPO11 alongside dozens of other proteins, enabling comprehensive profiling in heterogeneous cell populations like cancer samples.

• Proximity labeling proteomics: BioID or APEX2 fusions with IPO11 combined with antibody validation could map the complete interactome of IPO11 under various conditions.

• Single-cell Western blotting: Combining with IPO11 antibodies could reveal cell-to-cell variation in IPO11 expression and correlate with cellular phenotypes.

• CRISPR screens with antibody-based readouts: High-throughput screens using IPO11 antibodies as readouts could identify genes that regulate IPO11 expression, localization, or function.

• Conformational antibodies: Developing antibodies that specifically recognize cargo-bound versus cargo-free IPO11 could provide insights into transport kinetics.

How might IPO11 studies contribute to therapeutic developments for diseases like AML?

IPO11 research has significant therapeutic implications, particularly for AML treatment:

• Targeting leukemic stem cells (LSCs):

  • IPO11 knockdown decreases growth, reduces engraftment potential, and induces differentiation of LSCs

  • Antibody-based screening assays could identify small molecule inhibitors of the IPO11-BZW1/2 axis

• Biomarker development:

  • IPO11 antibodies could be used to develop diagnostic or prognostic assays for AML, potentially identifying patients with high LSC activity

  • Expression levels of IPO11 and its cargo proteins might predict treatment response

• Combination therapy strategies:

  • Understanding IPO11-dependent pathways could reveal synergistic targets for combination therapies

  • Antibody-based assays could measure treatment effects on nuclear transport dynamics

• Selective targeting approaches:

  • Research using IPO11 antibodies could help identify cancer-specific vulnerabilities in the nuclear transport machinery

  • Differences in IPO11 cargo specificity between normal and malignant cells might offer therapeutic windows

• Developmental toxicity screening:

  • Given IPO11's essential role in embryonic development , antibody-based assays could help screen potential therapeutics for developmental toxicity

The emerging understanding of IPO11 in regulating stem cell transcription factors positions it as a potential target for addressing therapy resistance and relapse in AML and potentially other cancers with similar dependencies .

IPO11 Antibody Comparison Table