IPO4 Antibody

What is IPO4 and what is its functional significance in cellular processes?

IPO4 (Importin-4) is a nuclear transport receptor belonging to the importin β family that mediates the import of specific proteins into the nucleus. It functions by recognizing nuclear localization signals (NLS) in cargo substrates and facilitating their docking to the nuclear pore complex (NPC). The importin/substrate complex is subsequently translocated through the nuclear pore via an energy-dependent, Ran-regulated mechanism .

At the nucleoplasmic side of the NPC, Ran binds to importin, causing dissociation of the importin/substrate complex. Importin is then re-exported from the nucleus to the cytoplasm, where GTP hydrolysis releases Ran . IPO4 plays a crucial role in transporting histones H3 and H4 into the nucleus for chromatin assembly and has been implicated in gastric cancer progression and poor prognosis .

What types of IPO4 antibodies are available and how do they differ in research applications?

IPO4 antibodies are available in both polyclonal and monoclonal formats from various manufacturers:

The choice between monoclonal and polyclonal antibodies depends on the specific research requirements. Polyclonal antibodies often provide higher sensitivity by recognizing multiple epitopes, while monoclonal antibodies offer greater specificity and consistency .

What are the recommended dilutions for IPO4 antibody in different experimental applications?

Optimal dilutions vary by application and specific antibody product:

It is recommended to titrate the antibody in each testing system to obtain optimal results, as the ideal concentration may be sample-dependent . For Western blotting, starting with mid-range dilutions and adjusting based on signal strength is advisable.

How should I optimize Western blot protocols specifically for IPO4 detection?

When optimizing Western blot protocols for IPO4 detection, consider the following parameters:

• Sample preparation: IPO4 has been successfully detected in various cell lines including HeLa, A549, LNCaP, HEK-293, Jurkat, K-562, HL-60, and THP-1 cells .

• Protein loading: Since IPO4 is a relatively large protein (observed molecular weight: 118-120 kDa), use freshly prepared samples and avoid repeated freeze-thaw cycles to prevent degradation.

• Gel percentage: Use 8-10% SDS-PAGE gels for optimal separation of proteins in the 118 kDa range.

• Transfer conditions: For large proteins like IPO4, longer transfer times or lower voltage may be necessary for complete transfer to the membrane.

• Blocking: Use 5% non-fat milk or BSA in TBST as blocking buffer.

• Primary antibody incubation: Incubate with the appropriate dilution of IPO4 antibody (see recommended dilutions in section 1.3) overnight at 4°C for optimal results.

• Expected band size: Look for a band at approximately 118-120 kDa, which is the observed molecular weight of IPO4 .

• Positive controls: HeLa cells and human brain tissue have been validated as positive controls for Western blot .

What are the critical considerations for immunohistochemistry experiments with IPO4 antibody?

For successful immunohistochemistry (IHC) with IPO4 antibody, consider these critical factors:

• Antigen retrieval: The recommended method is TE buffer pH 9.0, though citrate buffer pH 6.0 can serve as an alternative. This step is crucial for exposing epitopes that may be masked during fixation .

• Positive tissue controls: Human stomach cancer tissue has been validated as a positive control for IHC with IPO4 antibody .

• Antibody dilution: Use dilutions ranging from 1:50-1:500 for polyclonal antibodies and 1:250-1:1000 for monoclonal antibodies .

• Detection system: Choose an appropriate detection system based on host species (rabbit or mouse) and experiment requirements.

• Counterstaining: Use hematoxylin for nuclear counterstaining to provide contrast and allow proper visualization of IPO4 localization.

• Negative controls: Include controls without primary antibody or with isotype control antibodies to assess non-specific binding.

• Expected staining pattern: Since IPO4 is a nuclear transport protein, expect predominant cytoplasmic staining with potential nuclear membrane accentuation.

How can I effectively use IPO4 antibodies in immunofluorescence experiments?

For optimal immunofluorescence (IF) staining with IPO4 antibody:

• Cell line selection: HepG2 and MCF-7 cells have been validated for positive IF detection of IPO4 .

• Fixation method: Use 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature. Methanol fixation may serve as an alternative if membrane permeabilization is required.

• Permeabilization: If using PFA fixation, permeabilize cells with 0.1-0.5% Triton X-100 in PBS for 10 minutes.

• Blocking: Block with 1-5% BSA or normal serum from the species of the secondary antibody for 30-60 minutes.

• Antibody dilution: Use dilutions ranging from 1:200-1:800 for both polyclonal and monoclonal IPO4 antibodies .

• Incubation conditions: Incubate with primary antibody overnight at 4°C or for 1-2 hours at room temperature.

• Nuclear counterstain: Use DAPI or Hoechst dyes to visualize nuclei and assess IPO4 localization relative to nuclear structures.

• Expected localization pattern: As a nuclear transport protein, IPO4 typically shows predominantly cytoplasmic distribution with potential enrichment at the nuclear envelope and partial nuclear localization.

How can I validate IPO4 antibody specificity using knockout/knockdown approaches?

Validation of IPO4 antibody specificity through knockout (KO) or knockdown (KD) approaches is essential for ensuring reliable experimental results:

• KO-validated antibodies: Consider using pre-validated antibodies like the KO-validated IPO4 Rabbit Polyclonal Antibody (CAB19902), which has been tested in knockout systems to confirm specificity .

• CRISPR-Cas9 knockout validation:

  • Generate IPO4 knockout cell lines using CRISPR-Cas9 technology

  • Perform Western blot analysis comparing wild-type and knockout cells

  • A specific antibody will show a band at the expected molecular weight (118-120 kDa) in wild-type cells but not in knockout cells

• siRNA/shRNA knockdown validation:

  • Transfect cells with IPO4-specific siRNA/shRNA and appropriate controls

  • Harvest cells 48-72 hours post-transfection

  • Confirm knockdown efficiency at the mRNA level using qRT-PCR

  • Perform Western blot to demonstrate reduced IPO4 protein levels with your antibody

• Published validation: According to search results, there are at least 3 publications demonstrating KD/KO validation for certain IPO4 antibodies .

• Signal specificity: In addition to band presence/absence, evaluate whether non-specific bands appear in your control samples.

What are the main challenges in detecting IPO4 and how can they be addressed?

Several challenges may arise when detecting IPO4, and researchers should be aware of potential solutions:

• High molecular weight detection issues:

  • IPO4's large size (118 kDa) can make complete transfer during Western blotting difficult

  • Use longer transfer times, lower percentage gels (8-10%), and optimize transfer buffer conditions

  • Consider wet transfer systems for large proteins instead of semi-dry methods

• Epitope masking in fixed tissues:

  • Proper antigen retrieval is crucial for IHC applications

  • For IPO4, TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative

  • Optimize retrieval time and temperature based on tissue type and fixation conditions

• Background signal:

  • Increase blocking time/concentration

  • Optimize antibody concentrations by titration

  • Include additional washing steps

  • For immunofluorescence, use confocal microscopy to reduce out-of-focus signal

• Reproducibility issues:

  • Use consistent lot numbers when possible

  • Include appropriate positive controls (e.g., HeLa cells for WB, human stomach cancer tissue for IHC)

  • Document exact protocols including buffer compositions, incubation times, and temperatures

• Antibody cross-reactivity:

  • Select antibodies validated for your specific species of interest

  • IPO4 antibodies from different manufacturers have been validated for human, mouse, and rat samples

  • Consider using monoclonal antibodies for higher specificity if cross-reactivity is a concern

How can I use IPO4 antibodies to study protein-protein interactions in nuclear transport pathways?

IPO4 antibodies can be valuable tools for investigating protein-protein interactions within nuclear transport pathways:

• Co-immunoprecipitation (Co-IP):

  • IPO4 antibodies have been validated for Co-IP applications

  • Use 2-5 μg of antibody per 1 mg of protein lysate

  • Identify protein binding partners involved in nuclear transport

  • Include appropriate controls (IgG control, input lysate)

  • Confirm interactions through reverse Co-IP when possible

• Proximity ligation assay (PLA):

  • Detect in situ protein-protein interactions with spatial resolution

  • Combine IPO4 antibody with antibodies against suspected interaction partners

  • Visualize interactions as fluorescent dots indicating molecular proximity

• Immunofluorescence co-localization:

  • Perform double immunofluorescence staining with IPO4 antibody and antibodies against potential interacting proteins

  • Analyze co-localization using confocal microscopy

  • Calculate correlation coefficients (e.g., Pearson's, Manders') to quantify co-localization

• FRET/BRET studies:

  • Use antibodies to validate FRET/BRET results for protein interactions

  • Confirm proximity relationships detected through fluorescence/bioluminescence resonance energy transfer

• Pull-down assays:

  • Use IPO4 antibodies in conjunction with tagged recombinant proteins

  • Analyze IPO4-cargo interactions under different conditions (e.g., with/without RanGTP)

• Fractionation studies:

  • Separate nuclear and cytoplasmic fractions

  • Detect IPO4 and interacting partners in different cellular compartments

  • Monitor changes in localization under different conditions

How has IPO4 been implicated in disease research, and what role do antibodies play in these studies?

IPO4 has been implicated in several disease contexts, with antibodies playing a crucial role in elucidating its involvement:

• Cancer research:

  • IPO4 has been reported to contribute to gastric cancer progression and poor prognosis

  • Antibodies enable immunohistochemical assessment of IPO4 expression in tumor tissues

  • Western blot analysis can quantify IPO4 expression levels across different cancer types

  • Positive IHC has been detected in human stomach cancer tissue

• Nuclear transport dysregulation:

  • As a nuclear transport receptor, IPO4 dysfunction may affect the transport of critical proteins

  • Antibodies allow researchers to study subcellular localization changes in disease states

  • Immunofluorescence techniques reveal alterations in transport dynamics

• Histone transport and chromatin assembly:

  • IPO4 transports histones H3 and H4 into the nucleus for chromatin assembly

  • Antibodies can help study this process and its potential dysregulation in diseases involving chromatin structure

• Methodological approaches:

  • Tissue microarrays using IPO4 antibodies for screening across multiple cancer tissues

  • Co-localization studies to identify changes in IPO4-cargo interactions in disease states

  • Quantitative analysis of IPO4 expression in patient samples compared to normal controls

What recent advances have been made in antibody technologies that enhance IPO4 research?

Recent technological advances have significantly improved antibody-based IPO4 research:

• Knockout validation:

  • KO-validated antibodies, such as the Assay Genie CAB19902, provide enhanced confidence in specificity

  • This validation approach addresses the common concern of antibody cross-reactivity

• Phage display libraries:

  • Deep mutational scanning phage display libraries have revolutionized antibody epitope mapping

  • This technology allows for high-resolution characterization of antibody-antigen interactions

  • Although not specific to IPO4 in the search results, this approach could potentially be applied to IPO4 antibodies

• Active learning algorithms:

  • Recent research published in 2025 describes novel active learning strategies for improving antibody-antigen binding prediction

  • These computational approaches could enhance the development and validation of more specific IPO4 antibodies

• Multi-parameter analysis:

  • Combined approaches such as antibody library-on-library screening

  • Machine learning models for predicting antibody-antigen interactions

  • These methods could be adapted for IPO4 research to better understand binding specificity

• Recombinant antibody technology:

  • Production of recombinant antibody fragments with potentially improved binding properties

  • Engineering antibodies with enhanced specificity for particular IPO4 epitopes

  • Humanized antibodies for potential therapeutic applications

How can researchers effectively compare and integrate results from different IPO4 antibodies in their studies?

Effective comparison and integration of results from different IPO4 antibodies require careful consideration of several factors:

• Epitope mapping and antibody characteristics:

  • Document the specific epitopes recognized by each antibody

  • For instance, the Assay Genie CAB15600 targets amino acids 728-1081 of human IPO4

  • Different antibodies may recognize distinct regions of IPO4, potentially yielding complementary information

• Antibody validation metrics:

  • Create a standardized validation table for all antibodies used:

  Antibody ID	Type	Host	Epitope	Validated Applications	Positive Controls	KO/KD Validation	Cross-Reactivity
  11679-1-AP	Poly	Rabbit	Fusion protein Ag2281	WB, IHC, IF/ICC, IP, CoIP	HeLa cells, human brain	Yes (3 publications)	Human, mouse, rat
  67549-1-Ig	Mono	Mouse	Fusion protein Ag2281	WB, IHC, IF/ICC	A549, LNCaP, HeLa cells	Not specified	Human
  CAB19902	Poly	Rabbit	aa 728-1081	WB, ELISA	293T	KO validated	Human, mouse, rat

• Parallel validation experiments:

  • Run side-by-side comparisons using multiple antibodies on the same samples

  • Document concordant and discordant results

  • Investigate discrepancies to determine whether they represent technical issues or true biological differences

• Integration strategies:

  • Use multiple antibodies targeting different epitopes to confirm findings

  • Combine monoclonal and polyclonal approaches for complementary information

  • Validate key findings with at least two independent antibodies

  • Consider using antibodies from different host species for co-localization studies

• Data normalization and reporting:

  • Standardize quantification methods across different antibodies

  • Report antibody details explicitly in publications, including catalog numbers and dilutions

  • Include appropriate positive and negative controls for each antibody

  • Document lot numbers used, as antibody performance can vary between lots

What emerging research areas could benefit from improved IPO4 antibody technologies?

Several emerging research areas could significantly benefit from advances in IPO4 antibody technologies:

• Single-cell protein analysis:

  • Development of highly sensitive IPO4 antibodies compatible with single-cell Western blotting or CyTOF

  • Investigation of cell-to-cell variability in IPO4 expression and localization

  • Single-cell spatial proteomics to understand IPO4 distribution in heterogeneous tissues

• Live-cell imaging of nuclear transport:

  • Creation of cell-permeable IPO4 antibody fragments or nanobodies

  • Real-time visualization of IPO4-mediated cargo transport

  • Dynamic studies of IPO4 trafficking under different cellular conditions

• Therapeutic applications:

  • Development of antibodies that can modulate IPO4 function

  • Targeted approaches for diseases with aberrant nuclear transport

  • Combination with emerging drug delivery technologies

• Structural biology integration:

  • Antibodies that recognize specific conformational states of IPO4

  • Integration with cryo-EM studies to understand IPO4-cargo complex structures

  • Structure-function relationship studies of IPO4 in nuclear transport

• Systems biology approaches:

  • High-throughput screening of IPO4 cargo proteins using antibody-based techniques

  • Network analysis of IPO4-dependent nuclear transport pathways

  • Integration with multi-omics datasets to understand IPO4 regulation

How can researchers contribute to improving antibody validation standards for IPO4 research?

Researchers can contribute to improving validation standards for IPO4 antibodies through several approaches:

• Comprehensive validation reporting:

  • Document and publish detailed validation procedures

  • Include negative controls (IPO4 knockout or knockdown samples)

  • Share raw validation data in public repositories

  • Report both successful and unsuccessful antibody applications

• Cross-laboratory validation:

  • Establish collaborative networks for antibody validation

  • Compare antibody performance across different laboratory settings

  • Develop consensus protocols for IPO4 detection in various applications

• Integration of multiple validation methods:

  • Combine genetic approaches (CRISPR knockout, siRNA) with antibody testing

  • Use orthogonal methods to confirm antibody specificity

  • Implement both Western blot and immunofluorescence validation

  • Consider mass spectrometry validation of immunoprecipitated proteins

• Development of standardized positive controls:

  • Create reference cell lines with defined IPO4 expression levels

  • Develop recombinant IPO4 standards for quantitative applications

  • Share well-characterized positive control tissues or cell lines

• Community resources and databases:

  • Contribute to antibody validation databases like Antibodypedia

  • Provide detailed feedback to antibody manufacturers

  • Participate in community-wide antibody validation initiatives