KPNB1 Antibody, FITC conjugated

What is KPNB1 and what are its main cellular functions?

KPNB1 (Importin β1 or Karyopherin-β1) is a nucleocytoplasmic transport factor that plays critical roles in both cytoplasmic and nucleocytoplasmic transport mechanisms. It functions as a key component of the classical nuclear protein import pathway, where it transports cargo proteins into the nucleus either in conjunction with adaptor proteins like importin-α (KPNA) or directly in the non-classical pathway . Beyond nuclear transport, KPNB1 has specific cytoplasmic functions in cell size sensing and neuronal injury signaling that are distinct from its transport activity .

Recent research has significantly expanded our understanding of KPNB1's interactome to include various cytoplasmic and signaling complexes, highlighting potential new functions for the importin complex beyond nucleocytoplasmic transport . The multifunctionality of KPNB1 makes it an important protein in both normal cellular physiology and disease states, particularly in cancer where its expression is often dysregulated . Understanding these diverse functions requires specific tools like antibodies that can detect KPNB1 in different cellular compartments and experimental contexts.

Why would researchers use a FITC-conjugated KPNB1 antibody?

This direct visualization approach is particularly valuable for multicolor imaging experiments where researchers need to simultaneously examine KPNB1 alongside other cellular proteins. The bright green fluorescence of FITC allows clear visualization of KPNB1's subcellular distribution patterns, which is crucial given its dynamic shuttling between nuclear and cytoplasmic compartments . Additionally, FITC-conjugated antibodies are ideal for techniques like flow cytometry where direct labeling improves signal consistency and quantification accuracy when measuring KPNB1 expression levels across different cell populations or experimental conditions.

What applications are KPNB1 antibodies typically used for?

KPNB1 antibodies find utility across a wide range of research applications. In immunofluorescence microscopy, they enable visualization of KPNB1's subcellular localization, which typically shows both nuclear and cytoplasmic distribution with enrichment at the nuclear envelope . For Western blotting, KPNB1 antibodies detect the expected ~97 kDa protein band, allowing quantification of expression levels across different experimental conditions or tissue types .

These antibodies are also valuable for immunoprecipitation experiments to isolate KPNB1 complexes and identify interaction partners . The proximity ligation assay (PLA) represents another powerful application, allowing in situ visualization of protein-protein interactions with KPNB1 at single-molecule resolution . In cancer research, KPNB1 antibodies help investigate its role in malignancy, as elevated KPNB1 expression correlates with poor patient outcomes in head and neck squamous cell carcinoma and other cancers .

More specialized applications include biotinylation by antibody recognition (BAR) analysis, which has revealed numerous new interactors of importin β1, expanding the known KPNB1 interactome to include cytoplasmic and signaling complexes beyond its nuclear transport role . Flow cytometry with FITC-conjugated KPNB1 antibodies enables high-throughput quantitative analysis of expression levels in heterogeneous cell populations.

How do I determine the optimal dilution for KPNB1 antibody staining?

Determining the optimal dilution for KPNB1 antibody staining requires empirical testing for each specific application and experimental system. Based on published research, dilutions vary significantly depending on the application: from 1:50 for proximity ligation assays to 1:5000 for Western blotting . For immunofluorescence with FITC-conjugated antibodies, a reasonable starting point would be the manufacturer's recommended dilution, followed by a titration series (e.g., 1:100, 1:200, 1:500) to identify the optimal signal-to-noise ratio.

When optimizing dilutions, consider that some KPNB1 antibodies show compartment-specific recognition. For example, the monoclonal antibody described in the literature recognizes an epitope comprising residues 301-320 of human KPNB1 and demonstrates high specificity for cytoplasmic KPNB1 with minimal nuclear reactivity . This differential recognition may necessitate different optimal dilutions depending on whether you're examining predominantly cytoplasmic or nuclear KPNB1.

What cellular localization pattern should I expect when using a KPNB1 antibody?

How can I differentiate between cytoplasmic and nuclear KPNB1 using antibody-based techniques?

Differentiating between cytoplasmic and nuclear KPNB1 populations requires careful consideration of antibody characteristics and experimental methodology. Some antibodies demonstrate inherent compartment specificity - notably, a monoclonal antibody has been developed that targets residues 301-320 of human KPNB1 and shows remarkable specificity for cytoplasmic KPNB1 with minimal recognition of nuclear KPNB1 . This compartment-specific antibody provides a powerful tool for selectively examining cytoplasmic KPNB1 complexes.

For comprehensive analysis using standard antibodies, subcellular fractionation followed by Western blotting offers quantitative assessment of KPNB1 distribution. This approach requires careful validation of fractionation purity using compartment-specific markers (e.g., lamin for nuclear fraction, GAPDH for cytoplasmic fraction). Alternatively, high-resolution confocal microscopy with z-stack acquisition enables three-dimensional visualization of KPNB1 distribution. Nuclear counterstaining with DAPI provides a reference for defining nuclear boundaries, while quantitative image analysis measuring fluorescence intensities in nuclear versus cytoplasmic regions yields objective metrics of distribution .

How can KPNB1 antibodies help investigate its role in cancer progression?

KPNB1 antibodies serve as essential tools for investigating its role in cancer progression through multiple experimental approaches. Analysis of KPNB1 expression in tumor samples using immunohistochemistry or Western blotting with specific antibodies can reveal clinically relevant correlations. Data from The Cancer Genome Atlas (TCGA) demonstrates that KPNB1 expression is significantly elevated in head and neck squamous cell carcinoma (HNSCC) compared to normal tissue, and high expression correlates with poorer patient outcomes . KPNB1 antibodies enable validation of these findings in independent patient cohorts and experimental models.

Functionally, knockdown studies have demonstrated that KPNB1 is crucial for cancer cell survival. When KPNB1 was silenced using siRNA, researchers observed dramatically decreased clonogenic potential and enhanced apoptosis in HNSCC cell lines . Antibodies are essential for confirming successful knockdown at the protein level and for examining downstream effects on nuclear transport and other KPNB1-dependent processes. Additionally, KPNB1 inhibition using the small molecule importazole (IPZ) enhances radiation-induced apoptosis and radiosensitivity in cancer cells, suggesting therapeutic potential .

Mechanistically, KPNB1 antibodies help elucidate how this transport factor contributes to cancer hallmarks. For instance, IPZ treatment elevates PUMA (p53-upregulated modulator of apoptosis) expression by blocking the nuclear import of ΔNp63 in HNSCC cells . Furthermore, KPNB1 blockade attenuates radiation-induced upregulation of cell surface PD-L1 expression, identifying a potential link to immune evasion mechanisms . Antibody-based detection of these molecular changes provides critical insights into how KPNB1 functions as a pro-oncogenic factor and potential therapeutic target.

What considerations should be made when using KPNB1 antibodies in co-localization studies?

Co-localization studies with KPNB1 antibodies require careful experimental design to ensure reliable results. When using FITC-conjugated KPNB1 antibodies, select complementary fluorophores with minimal spectral overlap to prevent bleed-through artifacts. FITC's emission spectrum (peak ~515 nm) makes far-red fluorophores like Cy5 or Alexa 647 ideal partners, while yellow-orange fluorophores may require careful compensation .

Fixation and permeabilization conditions must be optimized to maintain epitope accessibility for all antibodies in the multiplexing panel. Different fixatives (paraformaldehyde, methanol, or acetone) can preferentially preserve certain epitopes while masking others, potentially creating artificial co-localization patterns. Since KPNB1 functions in dynamic complexes with numerous proteins including importin-α, Ran-GTP, and various cargoes, consider that standard co-localization analysis may not capture transient interactions .

For detecting such transient interactions, proximity ligation assays (PLA) offer higher sensitivity by generating fluorescent signals only when proteins are within 40 nm of each other . This approach has been successfully used with KPNB1 antibodies at dilutions as low as 1:50 to detect specific interaction partners . For quantitative co-localization analysis, include appropriate controls: single-stained samples to assess bleed-through, non-interacting protein pairs as negative controls, and known KPNB1 interaction partners as positive controls.

Super-resolution microscopy techniques can overcome the diffraction limit of conventional microscopy (approximately 200-250 nm), providing more accurate assessment of spatial relationships between KPNB1 and potential interaction partners. This is particularly important at the nuclear envelope, where multiple transport factors concentrate in close proximity but may form distinct functional complexes.

How do different fixation methods affect KPNB1 epitope recognition by antibodies?

The differential recognition of nuclear versus cytoplasmic KPNB1 observed with some antibodies suggests that fixation conditions may influence epitope accessibility in different cellular compartments . The monoclonal antibody described in the literature, which recognizes residues 301-320 of KPNB1, demonstrates strong specificity for cytoplasmic KPNB1 with minimal nuclear reactivity across diverse applications . This compartment-specific recognition could reflect differences in KPNB1 conformation, protein-protein interactions, or post-translational modifications between these compartments that are preserved or altered by specific fixation protocols.

When developing immunofluorescence protocols using KPNB1 antibodies, systematic comparison of fixation methods is recommended: standard PFA (2-4%), glutaraldehyde-enhanced PFA for stronger fixation, methanol, acetone, or combinations like methanol-acetone. The optimal protocol will depend on the specific antibody clone, experimental goals, and cell type being studied. For FITC-conjugated antibodies, remember that some fixatives can increase cellular autofluorescence in the green channel, potentially reducing signal-to-noise ratio.

How can I validate KPNB1 antibody specificity in knockdown experiments?

Validating KPNB1 antibody specificity through knockdown experiments is essential for ensuring reliable research outcomes. Begin by establishing effective KPNB1 silencing using siRNA transfection, a method that has been successfully employed in published studies where KPNB1 knockdown significantly decreased clonogenic potential and enhanced apoptosis in cancer cells . For comprehensive validation, analyze control and KPNB1-knockdown samples using multiple detection methods to confirm consistent signal reduction.

Western blotting represents a primary validation approach, where KPNB1 antibodies should detect a specific band at approximately 97 kDa that shows substantial reduction in knockdown samples compared to controls. Include appropriate loading controls such as GAPDH or β-actin to confirm equal protein loading . Immunofluorescence microscopy with the FITC-conjugated KPNB1 antibody should demonstrate markedly reduced staining intensity in knockdown cells compared to controls when imaged under identical acquisition settings. This visual confirmation provides spatial information about knockdown efficiency and confirms the specificity of fluorescence signals.

Flow cytometry offers quantitative assessment of signal reduction across cell populations, helping to account for transfection heterogeneity. For all methods, the degree of signal reduction should correlate with knockdown efficiency as measured by RT-qPCR. Additionally, include a non-targeting siRNA control to rule out non-specific effects of the transfection process. For partial knockdowns, examine dose-dependent relationships between KPNB1 mRNA levels, protein levels detected by antibodies, and biological phenotypes to further validate specificity.

What is the recommended protocol for immunofluorescence staining using FITC-conjugated KPNB1 antibodies?

For optimal immunofluorescence staining using FITC-conjugated KPNB1 antibodies, the following detailed protocol is recommended based on published methodologies:

Day 1:

• Culture cells on glass coverslips to 70-80% confluence

• Wash cells twice with PBS at room temperature

• Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature

• Wash three times with PBS, 5 minutes each

• Permeabilize cells with 0.2% Triton X-100 in PBS for 10 minutes

• Block with 5% normal serum and 1% BSA in PBS for 1 hour at room temperature

• Incubate with FITC-conjugated KPNB1 antibody diluted in blocking buffer (recommended starting concentration of 25 μg/mL based on published protocols) for 3 hours at room temperature or overnight at 4°C in a humidified chamber

Day 2:
8. Wash four times with PBS, 5 minutes each
9. Counterstain nuclei with DAPI (1 μg/mL in PBS) for 5 minutes
10. Wash twice with PBS, 5 minutes each
11. Mount coverslips using anti-fade mounting medium
12. Seal edges with nail polish and store at 4°C protected from light

For optimal results, include appropriate controls in each experiment. A negative control omitting the primary antibody will help establish the level of background autofluorescence and non-specific binding. When imaging, be mindful of FITC's susceptibility to photobleaching and adjust exposure settings accordingly. KPNB1 localization should be visible in both the cytoplasm and nucleus, with particular enrichment in the nuclear envelope region, though some antibodies may show preferential staining of cytoplasmic KPNB1 .

How can I use FITC-conjugated KPNB1 antibodies to identify novel interaction partners?

FITC-conjugated KPNB1 antibodies can be powerful tools for identifying novel interaction partners through several complementary approaches. Proximity ligation assays (PLA) offer a highly sensitive method for detecting protein-protein interactions in situ with single-molecule resolution. For PLA applications, researchers have successfully used KPNB1 antibodies at dilutions as low as 1:50 . This approach involves using the FITC-conjugated KPNB1 antibody alongside antibodies against potential interaction partners, followed by detection with secondary antibodies linked to oligonucleotides that, when in close proximity, enable rolling circle amplification and fluorescent probe hybridization.

Biotinylation by antibody recognition (BAR) represents another powerful approach specifically developed for KPNB1 interactome analysis . This method uses antibody-guided deposition of horseradish peroxidase (HRP) to generate tyramide radicals that biotinylate proteins in close proximity to KPNB1. The resulting biotinylated proteins can be purified using streptavidin beads and identified by mass spectrometry. This approach has revealed numerous new interactors of importin β1, expanding the KPNB1 interactome to include cytoplasmic and signaling complexes that highlight potential functions beyond nucleocytoplasmic transport .

For co-immunoprecipitation approaches, FITC-conjugated antibodies can be captured using anti-FITC antibodies coupled to beads, pulling down KPNB1 along with its interaction partners. Additionally, fluorescence resonance energy transfer (FRET) measurements between FITC on the KPNB1 antibody and acceptor fluorophores on antibodies against potential interaction partners can provide evidence of close molecular proximity. When validating novel interactions, combining multiple complementary techniques strengthens confidence in the findings and provides different perspectives on the nature and context of the interactions.

What is the best approach for quantifying KPNB1 nuclear-cytoplasmic ratios using fluorescence microscopy?

Quantifying KPNB1 nuclear-cytoplasmic ratios requires systematic image acquisition and analysis methodology. Based on approaches used in nuclear transport studies, the following comprehensive workflow is recommended:

For sample preparation and imaging, maintain consistent fixation and permeabilization conditions across all experimental samples to avoid artifacts in compartment-specific staining. Include nuclear counterstain (DAPI) for definitive nuclear segmentation . Acquire confocal microscopy images with sufficient resolution to clearly distinguish nuclear and cytoplasmic compartments, ensuring imaging parameters are within the linear range of detection and below saturation. For statistical robustness, collect multiple fields (minimum 10-15) per experimental condition.

The image analysis workflow should begin with nuclear segmentation using the DAPI channel to create a binary nuclear mask. Define the cytoplasmic region either by creating a ring around the nucleus (typically 5-10 pixels width) or by using a cytoplasmic marker. Measure mean fluorescence intensity of KPNB1 antibody staining within both the nuclear and cytoplasmic regions for each cell, then calculate the nuclear-to-cytoplasmic ratio (N/C). For statistical validity, analyze a minimum of 50-100 cells per condition.

Data interpretation should include comparison of N/C ratios across experimental conditions with appropriate statistical testing. Consider classifying cells into categories based on ratio values (nuclear-predominant, cytoplasmic-predominant, or equal distribution) to identify shifts in population distributions. For specialized applications, consider using antibodies with compartment-specific recognition properties, such as those that preferentially detect cytoplasmic KPNB1 . Image analysis software packages like CellProfiler, ImageJ with appropriate plugins, or commercial platforms can automate this workflow for high-throughput analysis.

How should I optimize cell permeabilization conditions for KPNB1 antibody staining?

Optimizing cell permeabilization is critical for balancing antibody accessibility to KPNB1 epitopes while preserving cellular architecture. Different permeabilization methods can significantly affect staining patterns, especially for proteins like KPNB1 that shuttle between nuclear and cytoplasmic compartments. For systematic optimization, test multiple permeabilization conditions in parallel:

Detergent-based methods include Triton X-100 (0.1-0.5%), which provides strong permeabilization for access to nuclear and cytoplasmic epitopes; Saponin (0.1-0.5%), which creates smaller pores and better preserves membrane structures; and Tween-20 (0.1-0.2%), a mild detergent for minimal disruption of cellular architecture. Organic solvent methods include methanol (100%, -20°C, 10 minutes), which simultaneously fixes and permeabilizes and can expose different epitopes; and acetone (100%, -20°C, 5 minutes), which offers rapid permeabilization but may disrupt some epitopes. Combination approaches include fixing with PFA followed by detergent permeabilization (standard approach) or using methanol:acetone (1:1) for combined fixation/permeabilization.

The compartment-specific recognition observed with some KPNB1 antibodies suggests that permeabilization method can significantly influence detection patterns . The monoclonal antibody described in the literature preferentially detected cytoplasmic KPNB1, indicating that optimization should evaluate both nuclear and cytoplasmic staining patterns under different conditions . After testing different permeabilization methods, evaluate results based on signal intensity, signal-to-noise ratio, specificity (compared to negative controls), and preservation of expected subcellular localization patterns. For FITC-conjugated antibodies, be particularly attentive to potential increases in autofluorescence that certain permeabilization methods may cause in the green channel.

What are effective strategies for multiplex imaging with FITC-conjugated KPNB1 antibodies?

Multiplex imaging with FITC-conjugated KPNB1 antibodies requires careful planning to minimize spectral overlap and maximize information gain. For fluorophore selection and pairing, when using FITC (excitation/emission: 499/515 nm), choose spectrally distant fluorophores as partners . Recommended combinations include FITC + Cy3/TRITC + Cy5/APC or FITC + DAPI + Alexa 647. Avoid fluorophores with significant spectral overlap with FITC, such as YFP or Alexa 488, which would require complex compensation or unmixing.

A sequential imaging approach is often preferable, imaging fluorophores one after another rather than simultaneously when using filter sets with broader bandwidths. Start with longer wavelength fluorophores and progress to shorter wavelengths to minimize photobleaching effects on FITC, which is relatively susceptible to photobleaching. For optimal fixation and antibody dilutions, reference successful protocols in the literature, such as using 25 μg/mL for 3 hours at room temperature for KPNB1 antibodies .

For advanced multiplex techniques, consider spectral unmixing for fluorophores with partial overlap or cyclic immunofluorescence with antibody stripping and reprobing for highly complex multiplexing needs. Multispectral imaging systems can provide better separation of fluorophores with similar emission profiles. Always include comprehensive controls: single-stained samples for each fluorophore to assess bleed-through, biological controls to validate co-localization patterns, and antibody competition assays to confirm specificity in multiplex settings.

Published research demonstrates that KPNB1 staining can be successfully combined with NorthernLights 557-conjugated secondary antibodies and DAPI counterstain, validating compatibility in a three-color imaging approach . For analyzing multiplex images, consider both qualitative assessment of localization patterns and quantitative colocalization analysis using established metrics like Pearson's correlation coefficient or Manders' overlap coefficient.

How is KPNB1 expression altered in cancer cells and what are the implications for therapeutic targeting?

KPNB1 expression shows significant alterations in cancer cells with important therapeutic implications. Analysis of The Cancer Genome Atlas (TCGA) data revealed that KPNB1 expression levels in head and neck squamous cell carcinoma (HNSCC) tumor tissue were significantly higher than in normal tissue . Furthermore, Kaplan-Meier analysis demonstrated that HNSCC patients with high KPNB1 expression had poorer clinical outcomes . This pattern of KPNB1 overexpression has been documented across several cancer types and transformed cell lines, suggesting a broad role in oncogenesis .

Functionally, KPNB1 appears critical for cancer cell survival. Knockdown experiments using siRNA targeting KPNB1 dramatically decreased clonogenic potential of cancer cells to approximately 3% for SAS cells and 36% for Ca9-22 cells, while simultaneously enhancing apoptosis . These findings indicate that cancer cells may develop dependence on elevated KPNB1 levels, creating a potential therapeutic vulnerability. This hypothesis is further supported by studies of KPNB1 inhibitors, including a 2-aminothiazole derivative (compound 1) that demonstrates strong anti-proliferative effects against several cancer cell lines at nanomolar concentrations .

Mechanistically, KPNB1 inhibition affects cell cycle progression, with compound 1 treatment interfering with G2/M phase progression and inducing apoptosis through the intrinsic pathway . Fluorescence microscopic analysis revealed that treated cells predominantly exhibited mitotic abnormalities with monopolar spindles . Importantly, this effect was not due to disruption of microtubule polymerization, suggesting a specific mechanism related to KPNB1 function rather than generalized cytotoxicity . Together, these findings position KPNB1 as a promising target for cancer therapeutics development, with particular potential in tumors showing KPNB1 overexpression.

How does KPNB1 contribute to radiation resistance in cancer cells?

KPNB1 plays a significant role in radiation resistance of cancer cells through multiple mechanisms, making it a promising target for radiosensitization strategies. Research in head and neck squamous cell carcinoma (HNSCC) has demonstrated that blocking KPNB1 function, either through the specific inhibitor importazole (IPZ) or through siRNA-mediated knockdown, specifically enhanced radiation-induced apoptosis and radiosensitivity of cancer cells .

The molecular mechanism underlying this effect involves regulation of p53-upregulated modulator of apoptosis (PUMA) expression. Treatment with importazole elevated PUMA expression by blocking the nuclear import of SCC-specific oncogene ΔNp63 in HNSCC cells . This suggests that KPNB1 normally facilitates the nuclear translocation of factors that suppress pro-apoptotic pathways, thus contributing to radiation resistance. By inhibiting this transport function, cancer cells become more susceptible to radiation-induced cell death.

Additionally, KPNB1 blockade attenuated the upregulation of cell surface programmed death-ligand 1 (PD-L1) expression on irradiated HNSCC cells . This finding has important implications for combination therapies, as PD-L1 upregulation represents a mechanism by which irradiated cancer cells can evade immune surveillance. By preventing this upregulation, KPNB1 inhibition could potentially enhance both direct radiation-induced cytotoxicity and immune-mediated tumor cell killing.

Importantly, the cytotoxic effect of importazole was less pronounced in normal human umbilical vein endothelial cells (HUVEC) compared to HNSCC cells, and importazole hardly enhanced radiation-induced apoptosis in HUVEC . This differential effect suggests a potential therapeutic window for combining KPNB1 inhibition with radiation therapy to enhance tumor control while minimizing normal tissue toxicity.

What methods are available for studying the KPNB1 interactome?

Studying the KPNB1 interactome has revealed expanding roles for this transport factor beyond classical nucleocytoplasmic transport. Several complementary methodologies have been developed, each with distinct advantages for interactome analysis:

The BioID approach, which uses proximity-based biotinylation with N- and C-terminal fusions of KPNB1 with modified biotin ligase BirA, initially showed limited efficiency in detecting importin complex cargos . This limitation prompted the development of alternative approaches for more comprehensive interactome characterization. Biotinylation by Antibody Recognition (BAR) represents a significant methodological advancement specifically developed for KPNB1 interactome analysis . This technique utilizes a highly specific monoclonal antibody against KPNB1 to guide horseradish peroxidase (HRP) enzyme deposition, generating tyramide radicals that biotinylate proteins in close proximity to KPNB1. Importantly, the generated antibody recognized an epitope comprising residues 301-320 of human KPNB1 and showed high specificity for cytoplasmic KPNB1 complexes with minimal nuclear reactivity .

Proximity Ligation Assay (PLA) offers another approach for detecting specific KPNB1 interactions in situ . This method provides single-molecule resolution visualization of protein-protein interactions and has been successfully implemented with KPNB1 antibodies. Traditional co-immunoprecipitation (IP) followed by mass spectrometry remains valuable for identifying stable interaction partners, while crosslinking immunoprecipitation can capture more transient interactions.

These methodologies have led to significant discoveries about the KPNB1 interactome. BAR analysis with the cytoplasmic-specific KPNB1 antibody revealed numerous new interactors, expanding the known KPNB1 interactome to include cytoplasmic and signaling complexes . These findings highlight potential new functions for the importin complex beyond nucleocytoplasmic transport, including roles in cell size sensing and neuronal injury signaling . The comprehensive interactome data has been made publicly available via ProteomeXchange with identifier PXD032728, enabling further analysis by the research community .