KPNA4 Antibody, HRP conjugated

What is KPNA4 and what cellular functions does it serve?

KPNA4, also known as importin subunit alpha-3 or karyopherin-alpha4, is a cytoplasmic protein that recognizes nuclear localization signals (NLS) in cargo proteins and facilitates their transport to the nucleus . It functions as part of the classical nuclear import pathway and interacts with the NLSs of various proteins including DNA helicase Q1 and SV40 T antigen . With a molecular weight of approximately 58 kDa, KPNA4 plays a critical role in regulating protein trafficking between the cytoplasm and nucleus, which is essential for various cellular processes including gene expression, cell division, and signal transduction.

What are the recommended applications for KPNA4 antibodies?

KPNA4 antibodies have been validated for multiple experimental applications, with specific dilution recommendations for optimal results:

When using commercially available antibodies, it's critical to titrate the antibody in your specific testing system to determine the optimal concentration for your experimental conditions . For Western blotting applications, some manufacturers have specifically validated a 1:1000 dilution for optimal results .

How should KPNA4 antibodies be stored to maintain reactivity?

Most KPNA4 antibodies should be stored at -20°C and remain stable for one year after shipment . For antibodies in liquid form, they are typically supplied in preservative buffers such as PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) or TRIS buffered saline with 0.09% Sodium Azide and 0.5% Bovine Serum Albumin . Importantly, for many formulations, aliquoting is unnecessary for -20°C storage, which simplifies laboratory protocols . Smaller volume preparations (e.g., 20μl sizes) may contain additional stabilizers such as 0.1% BSA . Avoid repeated freeze-thaw cycles to maintain antibody integrity and performance.

What species reactivity can be expected with KPNA4 antibodies?

KPNA4 antibodies show varying cross-reactivity depending on the specific product. Based on current commercially available options:

When selecting an antibody for your research, verify the validated species reactivity from the manufacturer and consider that antibody reactivity and working conditions may vary between species . This is especially important for comparative studies across multiple model organisms.

How can KPNA4 expression be correlated with immune cell infiltration in cancer tissues?

Recent research has revealed that KPNA4 expression levels positively correlate with immune cell infiltration in hepatocellular carcinoma (HCC) . Specifically, the levels of infiltration of CD8+ T cells, B cells, dendritic cells, CD4+ T cells, neutrophils, and macrophages all show positive associations with KPNA4 expression levels . This correlation can be investigated using immunohistochemistry combined with computational approaches such as those available in the TIMER database.

Multivariable Cox regression analysis has determined that tumor stage, macrophage and dendritic cell levels, and KPNA4 expression are all significant risk factors for poor prognosis in HCC patients, with KPNA4 expression serving as an independent factor influencing 3-year and 5-year survival rates . These findings suggest that KPNA4 may play an important role in the tumor immune microenvironment and could potentially serve as a target for immunotherapy approaches.

What methodological considerations are important when using HRP-conjugated antibodies in KPNA4 detection systems?

When using HRP-conjugated detection systems for KPNA4, several methodological considerations are critical:

For ELISA applications, a sandwich enzyme-linked immunosorbent assay approach is commonly employed, where anti-KPNA4 antibody is pre-coated onto a 96-well plate, and biotin-conjugated anti-KPNA4 antibody serves as the detection antibody . After sample incubation and washing of unbound conjugates, HRP-Streptavidin is added to bind to the biotinylated detection antibody . Following a third washing step, TMB substrates are added to visualize the HRP enzymatic reaction, which produces a blue color that turns yellow after adding a stop solution . The optical density is then measured at 450nm in a microplate reader.

For Western blot applications with HRP-conjugated secondary antibodies, the optimal dilution of the primary KPNA4 antibody (typically 1:1000) must be determined empirically . The choice of blocking buffer, incubation time, and washing conditions can significantly impact the signal-to-noise ratio. Additionally, the detection method (chemiluminescence vs. chromogenic) must be selected based on the required sensitivity and available equipment.

How can researchers differentiate between specific and non-specific binding when using KPNA4 antibodies?

Distinguishing specific from non-specific binding is crucial for accurate data interpretation in KPNA4 research. Several validation approaches are recommended:

• Molecular weight verification: KPNA4 should appear at approximately 58 kDa on Western blots, which matches both its calculated and observed molecular weight . Any significant deviation from this expected size may indicate non-specific binding or post-translational modifications.

• Positive and negative controls: Include known positive samples (e.g., A549 cells, NIH/3T3 cells, HeLa cells, or Jurkat cells) as well as negative controls . KPNA4 knockdown or knockout samples provide excellent negative controls for verifying antibody specificity.

• Cross-validation with multiple antibodies: Using different antibodies that recognize distinct epitopes of KPNA4 can help confirm specific binding. If multiple antibodies show consistent results, this increases confidence in the specificity of the detected signal.

• Competition assays: Pre-incubating the antibody with the immunizing peptide (if available) before application to the sample should abolish specific binding signals while leaving non-specific binding intact.

What is the relationship between KPNA4 expression and T cell exhaustion markers in cancer research?

KPNA4 expression has been found to correlate with T cell exhaustion markers in hepatocellular carcinoma, suggesting a potential role in immunosuppression . Specifically, positive correlations have been observed between KPNA4 expression and CTLA4, GZMB, and PDL1 (CD274) expressions in cancerous tissues . These relationships suggest that KPNA4 may be involved in regulating the immune checkpoint pathway.

The correlation with these immune checkpoint molecules makes KPNA4 a potential target for cancer immunotherapy research. Researchers investigating this relationship should consider:

• Using multiplex immunohistochemistry or flow cytometry to simultaneously detect KPNA4 and T cell exhaustion markers in tumor samples

• Employing KPNA4 knockdown or overexpression models to evaluate the causal relationship with immune checkpoint expression

• Analyzing patient cohorts receiving immune checkpoint inhibitor therapy to determine if KPNA4 expression correlates with treatment response

How can optimal signal-to-noise ratio be achieved in Western blot applications with KPNA4 antibodies?

Optimizing Western blot protocols for KPNA4 detection requires careful attention to several parameters:

• Antibody dilution optimization: While manufacturers recommend dilutions ranging from 1:1000 to 1:8000 for KPNA4 antibodies in Western blot applications , the optimal concentration should be empirically determined for each experimental system. Start with the recommended dilution and adjust based on signal strength and background.

• Blocking optimization: Insufficient blocking is a common cause of high background. Test different blocking agents (e.g., BSA, non-fat dry milk, commercial blocking buffers) to determine which provides the best signal-to-noise ratio for your specific antibody.

• Washing protocol: Thorough washing between steps is critical. Typically, TBST (TBS with 0.05-0.1% Tween-20) is used, with at least three 5-10 minute washes after each antibody incubation step.

• Incubation time and temperature: Optimizing incubation conditions can significantly impact results. For primary antibodies, overnight incubation at 4°C often provides better specificity than shorter incubations at room temperature.

• Sample preparation: Ensure complete protein denaturation and use fresh samples when possible to avoid protein degradation that can lead to non-specific bands.

What strategies can address inconsistent results in immunohistochemical detection of KPNA4?

Inconsistent immunohistochemical results when detecting KPNA4 in tissue samples may be addressed through the following approaches:

• Antigen retrieval optimization: For KPNA4 detection, antigen retrieval with TE buffer at pH 9.0 is suggested, although citrate buffer at pH 6.0 can also be used as an alternative . Compare both methods to determine which works best for your specific tissue samples.

• Fixation considerations: Overfixation can mask epitopes, while underfixation may lead to poor tissue morphology. Standardize fixation protocols (duration, fixative composition) across all samples to improve consistency.

• Section thickness standardization: Maintain consistent section thickness (typically 4-5 μm) as variations can affect antibody penetration and staining intensity.

• Positive and negative controls: Include known positive tissues (e.g., human colon cancer tissue has been validated for KPNA4 detection ) and appropriate negative controls in each staining run.

• Automated vs. manual staining: Consider using automated staining platforms to reduce variability in incubation times, washing steps, and reagent application.

How can researchers quantitatively assess KPNA4 expression levels in clinical samples?

Quantitative assessment of KPNA4 expression in clinical samples requires standardized approaches:

• Western blot quantification: Use image analysis software to quantify band intensity, normalizing to loading controls such as GAPDH or β-actin. For reproducibility, include standard curves with known quantities of recombinant protein when possible.

• RT-qPCR for mRNA quantification: Design and validate specific primers for KPNA4, use appropriate reference genes, and follow MIQE guidelines for accurate quantification of transcript levels.

• ELISA-based quantification: ELISA kits for KPNA4 provide a quantitative approach for protein detection in biological fluids or tissue lysates . The sandwich ELISA format offers high specificity through the use of two antibodies recognizing different epitopes.

• Immunohistochemical scoring systems: Implement standardized scoring systems such as H-score or Allred score for semi-quantitative assessment of KPNA4 immunostaining. These systems typically combine intensity and percentage of positive cells.

• Digital pathology approaches: Utilize whole slide imaging and automated image analysis algorithms to objectively quantify KPNA4 expression in tissue sections, reducing inter-observer variability.

How does KPNA4 expression correlate with clinical parameters in hepatocellular carcinoma?

KPNA4 has demonstrated significant correlations with multiple clinical parameters in hepatocellular carcinoma (HCC):

• Expression levels: KPNA4 mRNA and protein expression levels are significantly higher in HCC samples compared to normal liver tissue . This differential expression has been validated across multiple datasets.

• Age correlation: KPNA4 expression levels tend to be higher in HCC patients over 21 years of age compared to younger healthy individuals .

• Tumor stage and grade: Increased KPNA4 mRNA expression is associated with higher tumor stage and grade in HCC patients, suggesting its potential role in disease progression .

• Survival correlation: KPNA4 expression is negatively correlated with patient survival (hazard ratio (HR) = 1.86, 95% confidence interval (CI): 1.29-2.7, P = 0.00076), indicating its potential value as a prognostic marker .

• Independent risk factor: Univariate and multivariate Cox regression analyses have identified KPNA4 as an independent risk factor for HCC patients .

These findings collectively suggest that KPNA4 may serve as both a diagnostic biomarker and prognostic indicator in HCC, potentially guiding treatment decisions and patient management strategies.

What methodological approaches can be used to investigate KPNA4's role in nuclear transport of disease-related proteins?

Investigating KPNA4's role in nuclear transport of disease-related proteins requires a multi-faceted approach:

• Co-immunoprecipitation (Co-IP) assays: KPNA4 antibodies have been validated for Co-IP applications, allowing researchers to identify protein-protein interactions between KPNA4 and potential cargo proteins . This approach can reveal novel transport substrates relevant to disease processes.

• Nuclear/cytoplasmic fractionation: Combined with Western blot analysis, this technique can assess how modulation of KPNA4 levels (via knockdown or overexpression) affects the subcellular distribution of cargo proteins.

• Immunofluorescence microscopy: Using validated antibodies for IF/ICC applications (recommended dilution 1:200-1:800) , researchers can visualize the co-localization of KPNA4 with cargo proteins during nuclear import processes.

• CRISPR/Cas9-mediated gene editing: Creating KPNA4 knockout or mutant cell lines can provide insights into the specificity of KPNA4-mediated transport compared to other karyopherin family members.

• In vitro binding assays: Using recombinant proteins to assess direct interactions between KPNA4 and nuclear localization signals (NLSs) of disease-associated proteins.

• Proximity ligation assays: This technique can visualize and quantify interactions between KPNA4 and cargo proteins with high sensitivity and specificity in situ.

How can KPNA4 be targeted for therapeutic development in cancer?

Given KPNA4's role as a potential biomarker and its correlation with poor prognosis in HCC, several therapeutic approaches could be explored:

• Small molecule inhibitors: Developing compounds that specifically disrupt KPNA4-cargo protein interactions could potentially inhibit the nuclear import of oncogenic proteins. This approach requires detailed structural information about KPNA4 binding pockets.

• Peptide-based inhibitors: Synthetic peptides mimicking natural nuclear localization signals (NLSs) could competitively inhibit KPNA4-mediated nuclear transport of cancer-promoting proteins.

• RNA interference strategies: siRNA or antisense oligonucleotides targeting KPNA4 mRNA could reduce its expression, potentially slowing cancer progression based on its established correlation with tumor stage and grade .

• Combination with immunotherapy: Given KPNA4's correlation with immune cell infiltration and T cell exhaustion markers like CTLA4 and PDL1 , combining KPNA4 inhibition with immune checkpoint blockade might enhance therapeutic efficacy.

• Antibody-drug conjugates: Developing antibody-drug conjugates targeting KPNA4 could deliver cytotoxic payloads specifically to cells overexpressing this protein.

Each of these approaches requires extensive validation in preclinical models before advancing to clinical testing, with careful attention to potential off-target effects given KPNA4's important physiological functions.

What novel detection methods might improve sensitivity and specificity for KPNA4 in complex biological samples?

Advancing KPNA4 detection technology could enhance both research and clinical applications:

• Multiplex protein detection platforms: Developing assays that simultaneously detect KPNA4 along with related transport proteins and cargo molecules could provide more comprehensive insights into nuclear transport dynamics in disease states.

• Mass spectrometry-based approaches: Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) could enable highly sensitive and specific quantification of KPNA4 in complex samples without reliance on antibodies.

• Proximity-based detection methods: Techniques such as proximity extension assay (PEA) or proximity ligation assay (PLA) could improve sensitivity for detecting KPNA4 protein-protein interactions in tissue samples.

• Single-cell analysis platforms: Developing methods to assess KPNA4 expression and function at the single-cell level could reveal heterogeneity within tumor microenvironments that might be masked in bulk tissue analysis.

• Aptamer-based detection: Developing specific aptamers against KPNA4 could provide alternative recognition molecules to antibodies, potentially offering advantages in certain applications.

These technological advances could not only improve KPNA4 detection sensitivity but also enable novel applications in both research and clinical settings, particularly for early disease detection and monitoring treatment response.