HNRNPAB Antibody, HRP conjugated

What is HNRNPAB and what are its primary functions in cellular biology?

HNRNPAB (Heterogeneous nuclear ribonucleoprotein A/B) is an RNA-binding protein that plays crucial roles in RNA metabolism and processing. It exhibits high affinity for G-rich and U-rich regions of heterogeneous nuclear RNA (hnRNA) and binds to APOB mRNA transcripts around RNA editing sites . The protein functions primarily in the nuclear metabolism of hnRNAs, especially for pre-mRNAs containing cytidine-rich sequences. HNRNPAB can also bind to single-stranded DNA with poly(C) sequences, suggesting its multifunctional nature in nucleic acid interactions. Recent research has demonstrated its involvement in various cellular processes including proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in certain cancer types .

What are the common applications for HNRNPAB antibodies in research?

HNRNPAB antibodies find utility across numerous experimental techniques and research applications:

Researchers typically use these antibodies to investigate HNRNPAB's role in cancer progression, RNA processing mechanisms, and as potential biomarkers for disease states .

What are the optimal conditions for using HRP-conjugated HNRNPAB antibodies in Western blotting?

For optimal Western blot results with HRP-conjugated HNRNPAB antibodies, researchers should consider the following protocol parameters:

• Sample preparation: Extract 25μg of protein per lane using standard lysis buffers containing protease inhibitors .

• Blocking conditions: Use 3% non-fat dry milk in TBST for 1 hour at room temperature to minimize background signal .

• Antibody dilution: A dilution of 1:1000 to 1:2000 typically yields optimal results for HRP-conjugated HNRNPAB antibodies .

• Incubation time: Incubate membranes with the antibody for 2-2.5 hours at room temperature or overnight at 4°C for enhanced sensitivity .

• Detection method: Employ ECL-based detection systems, with exposure times typically ranging from 30 seconds to 5 minutes depending on expression levels .

• Controls: Include appropriate positive controls (e.g., HeLa or brain tissue lysates) and negative controls (HNRNPAB knockout cell lysates where available) to validate specificity .

Researchers should note that reducing agents in sample buffers and membrane overexposure may occasionally affect signal quality with HRP-conjugated antibodies.

How should HRP-conjugated HNRNPAB antibodies be validated for specificity?

Thorough validation of HRP-conjugated HNRNPAB antibodies is essential to ensure experimental reliability and reproducibility:

• Knockout/knockdown validation: Test the antibody on samples from HNRNPAB knockout or knockdown models. For example, utilizing Human HNRNPAB knockout HEK-293T cell lines can confirm antibody specificity through the absence of signal in these samples .

• Multiple detection methods: Validate antibody performance across different techniques (WB, IHC, ICC) to ensure consistent recognition of the target protein.

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the antibody specifically pulls down HNRNPAB rather than related proteins with similar sequence homology.

• Cross-reactivity assessment: Test the antibody against closely related hnRNP family members (such as hnRNP A1, A2/B1, E1/PCBP1, K) to ensure specificity .

• Species cross-reactivity: Verify performance across human, mouse, and rat samples if multispecies applications are intended .

Proper validation not only confirms antibody specificity but also helps troubleshoot potential experimental issues and enhances data reproducibility.

What troubleshooting steps should be taken when HRP-conjugated HNRNPAB antibodies show weak or non-specific signals?

When encountering signal problems with HRP-conjugated HNRNPAB antibodies, consider these troubleshooting approaches:

For weak signals:

• Increase antibody concentration (try 1:500 instead of 1:1000)

• Extend incubation time (overnight at 4°C)

• Load more protein (up to 50μg)

• Use enhanced sensitivity detection substrates

• Check sample preparation method for potential protein degradation

• Optimize antigen retrieval for IHC/ICC applications

For non-specific signals:

• Use more stringent washing conditions (increase wash time or TBST concentration)

• Optimize blocking conditions (try 5% BSA instead of milk)

• Reduce antibody concentration

• Pre-adsorb antibody with knockout lysates

• Use fresh ECL reagents and avoid membrane overexposure

• Include appropriate loading controls (e.g., GAPDH antibody at 1:20000 dilution)

For false negatives, verify that the epitope hasn't been masked by post-translational modifications or protein interactions in your specific samples.

How can HRP-conjugated HNRNPAB antibodies be utilized to study its role in cancer progression?

HNRNPAB has emerged as a significant marker in various cancer types, particularly in non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). HRP-conjugated antibodies offer researchers powerful tools to investigate its oncogenic mechanisms:

• Expression profiling: Quantitative immunohistochemistry using HRP-conjugated antibodies can assess HNRNPAB overexpression in tumor tissues compared to adjacent normal tissues. Studies have shown that HNRNPAB is significantly upregulated in NSCLC tissues and correlates with poor prognosis, particularly in lung adenocarcinoma (LUAD) .

• Subcellular localization studies: Immunocytochemistry with HRP-conjugated antibodies can track changes in HNRNPAB's nuclear-cytoplasmic distribution during cancer progression. Research indicates increased cytoplasmic localization of HNRNPAB during the dedifferentiation of hepatocellular carcinoma, suggesting this localization pattern could serve as a potential diagnostic biomarker .

• Molecular interaction analysis: Co-immunoprecipitation using HRP-conjugated antibodies can identify HNRNPAB's interaction partners in tumorigenic pathways, particularly those involved in epithelial-mesenchymal transition (EMT).

• Therapeutic response monitoring: Western blotting with HRP-conjugated antibodies can assess changes in HNRNPAB expression following experimental treatments, potentially indicating efficacy of targeted therapies.

What methods can be used to study the interaction between HNRNPAB and nucleic acids?

Understanding HNRNPAB's interactions with RNA and DNA is crucial for elucidating its biological functions. Researchers can employ several approaches:

• RNA Immunoprecipitation (RIP): HRP-conjugated HNRNPAB antibodies can immunoprecipitate the protein along with its bound RNAs. Subsequent RT-qPCR or RNA sequencing can identify the specific RNA targets, with particular focus on G-rich and U-rich regions of hnRNA and APOB mRNA transcripts .

• Chromatin Immunoprecipitation (ChIP): Though primarily an RNA-binding protein, HNRNPAB can also interact with single-stranded DNA. ChIP using HRP-conjugated antibodies followed by sequencing can map these DNA interactions .

• Electrophoretic Mobility Shift Assays (EMSA): These assays can determine the binding affinity and specificity of HNRNPAB for different RNA sequences, complementing immunoprecipitation approaches.

• Fluorescence resonance energy transfer (FRET): This technique can visualize the dynamics of HNRNPAB-RNA interactions in living cells.

• Cross-linking and Immunoprecipitation (CLIP): This method helps identify the precise binding sites of HNRNPAB on its RNA targets at nucleotide resolution.

When designing nucleic acid interaction studies, researchers should consider HNRNPAB's documented preference for G-rich and U-rich regions, which may influence experimental design and interpretation .

How do post-translational modifications affect HNRNPAB detection with HRP-conjugated antibodies?

Post-translational modifications (PTMs) of HNRNPAB can significantly impact antibody recognition and may alter experimental outcomes when using HRP-conjugated antibodies:

• Phosphorylation: HNRNPAB contains multiple phosphorylation sites that may become modified during cellular signaling events. These modifications can either expose or mask antibody epitopes, potentially affecting detection sensitivity.

• Methylation: Arginine methylation of HNRNPAB can alter its RNA-binding properties and subcellular localization, which may influence antibody accessibility in certain experimental contexts.

• SUMOylation: Similar to related hnRNP family members like hnRNP K, HNRNPAB may undergo SUMOylation that affects its functional properties and potentially antibody recognition .

Researchers should consider employing modification-specific antibodies alongside total HNRNPAB antibodies to gain comprehensive insights into the protein's dynamic regulation. Additionally, phosphatase or desumoylase treatments prior to antibody incubation may reveal whether signal variations are due to PTMs rather than changes in total protein expression.

How does HNRNPAB expression correlate with clinical outcomes in cancer patients?

Extensive clinical studies have revealed significant correlations between HNRNPAB expression and patient outcomes:

These correlations position HNRNPAB as both a potential prognostic biomarker and therapeutic target, highlighting the importance of antibody-based detection methods in clinical research applications.

What experimental models are most appropriate for studying HNRNPAB function using HRP-conjugated antibodies?

Researchers investigating HNRNPAB function should select experimental models based on their specific research questions:

• Cell line models:

  • For cancer studies: NCI-H292 or PC-9 (NSCLC cell lines) have been validated for HNRNPAB knockdown studies

  • For basic mechanistic studies: HEK293T cells offer ease of transfection and manipulation

  • For antibody validation: HEK293T HNRNPAB knockout lines provide excellent negative controls

• Tissue models:

  • Human NSCLC tissues categorized by TNM classification allow correlation studies

  • Liver tissues from viral hepatitis and HCC patients enable evaluation of disease progression markers

  • Mouse brain tissue serves as a positive control for antibody validation

• Knockdown/knockout systems:

  • Lentiviral shRNA systems targeting HNRNPAB (using vectors like psi-LVRU6GP) can generate stable knockdown cell lines for functional studies

  • CRISPR/Cas9 knockout models provide complete ablation for stringent functional analysis

• Animal models:

  • Xenograft models using HNRNPAB-manipulated cells can assess in vivo relevance of in vitro findings

  • Transgenic mouse models with tissue-specific HNRNPAB alterations may reveal developmental and physiological roles

The choice between these models should be guided by the specific research question, available resources, and ethical considerations.

How can HNRNPAB detection be incorporated into multiparameter analysis of cancer tissues?

Integrating HNRNPAB detection into comprehensive cancer tissue analysis provides valuable insights into tumor biology and potential therapeutic strategies:

• Multiplex immunohistochemistry (mIHC):

  • HRP-conjugated HNRNPAB antibodies can be combined with antibodies against other cancer biomarkers using sequential staining protocols

  • This approach allows simultaneous visualization of HNRNPAB along with markers of proliferation (Ki-67), EMT (E-cadherin, vimentin), and cell cycle regulators

• Correlative microscopy:

  • Immunodetection of HNRNPAB can be aligned with RNA-FISH to simultaneously visualize protein localization and its RNA targets

  • This technique is particularly valuable for studying HNRNPAB's role in RNA processing and trafficking

• Integrated multi-omics:

  • HNRNPAB protein detection can be correlated with transcriptomic data from the same samples

  • The Linked Omics database has been used to screen genes associated with HNRNPAB expression in NSCLC, identifying potential downstream targets verified by RT-qPCR

• Liquid biopsy analysis:

  • Detection of circulating HNRNPAB in patient blood samples may provide minimally invasive biomarkers for cancer progression

  • Correlation with circulating tumor cells or cell-free DNA/RNA offers comprehensive cancer monitoring

These multiparameter approaches provide context for HNRNPAB's role in cancer biology and may reveal novel therapeutic targets within its regulatory network.

What are the key differences in detection sensitivity between various applications of HRP-conjugated HNRNPAB antibodies?

Different experimental techniques have varying sensitivity thresholds when using HRP-conjugated HNRNPAB antibodies:

When designing experiments, researchers should consider these sensitivity differences. For example, rare variants or low-abundance isoforms of HNRNPAB may require more sensitive techniques like ELISA, while subcellular localization studies benefit from the spatial resolution of ICC/IF despite potentially lower absolute sensitivity.

How should researchers optimize fixation and permeabilization conditions for immunocytochemistry with HRP-conjugated HNRNPAB antibodies?

Optimization of fixation and permeabilization is critical for successful immunocytochemistry with HNRNPAB antibodies:

• Fixation recommendations:

  • For nuclear localization studies: 4% paraformaldehyde (PFA) for 15 minutes at room temperature preserves nuclear architecture while maintaining epitope accessibility

  • For cytoplasmic detection: Methanol fixation (-20°C for 10 minutes) may provide better access to cytoplasmic epitopes

  • Avoid over-fixation, which can mask epitopes through excessive cross-linking

• Permeabilization considerations:

  • For balanced nuclear/cytoplasmic detection: 0.1-0.2% Triton X-100 for 10 minutes

  • For preferential nuclear detection: 0.5% Triton X-100 may enhance nuclear permeabilization

  • For cytoplasmic detection: 0.1% saponin provides gentler permeabilization that preserves cytoplasmic structures

• Protocol optimization:

  • Test multiple conditions to determine optimal parameters for your specific cell type

  • Include positive controls (cell lines with known HNRNPAB expression) and negative controls (HNRNPAB knockdown cells)

  • For dual localization studies, consider the subcellular distribution patterns observed in previous research, with HNRNPAB showing both nuclear prominence and cytoplasmic localization during cancer progression

These optimizations are particularly important when studying HNRNPAB's subcellular redistribution during cancer progression, as observed in hepatocellular carcinoma where cytoplasmic localization increases during dedifferentiation .

What considerations should be made when designing experiments to monitor dynamic changes in HNRNPAB expression?

When investigating dynamic changes in HNRNPAB expression or localization, researchers should address these key experimental design considerations:

• Time course analysis:

  • Select appropriate time points that capture the biological process of interest (e.g., cell cycle progression, differentiation, or response to treatment)

  • For cancer progression studies, include samples representing different stages of malignant transformation

  • Consider using live-cell imaging with fluorescently tagged HNRNPAB for real-time monitoring

• Sample preparation consistency:

  • Standardize protein extraction methods across all time points

  • Use identical fixation times and conditions for IHC/ICC applications

  • Process all samples in parallel to minimize batch effects

• Quantification methods:

  • Employ digital image analysis for immunohistochemistry to obtain objective quantification

  • Use appropriate housekeeping genes (e.g., GAPDH) as loading controls for Western blotting

  • Consider flow cytometry for single-cell quantification of HNRNPAB levels

• Experimental manipulations:

  • When conducting knockdown studies, verify knockdown efficiency at both mRNA and protein levels

  • For cancer studies, correlate HNRNPAB expression changes with functional readouts (proliferation, migration, invasion)

  • Include appropriate controls for treatments that might affect general protein synthesis or degradation

• Context-specific considerations:

  • Monitor both expression level and subcellular localization changes

  • Consider parallel analysis of HNRNPAB-associated genes identified through bioinformatics approaches

  • Assess post-translational modifications that might affect HNRNPAB function but not total protein levels

These methodological considerations ensure robust and reproducible assessment of HNRNPAB dynamics in complex biological systems.

What emerging technologies might enhance detection and analysis of HNRNPAB?

Several cutting-edge technologies show promise for advancing HNRNPAB research:

• Proximity ligation assays (PLA): These techniques could enable visualization of HNRNPAB interactions with specific RNA targets or protein partners in situ with single-molecule sensitivity.

• CRISPR-based tagging: Endogenous tagging of HNRNPAB using CRISPR/Cas9 allows for live-cell imaging of the native protein under physiological conditions, avoiding artifacts associated with overexpression.

• Single-cell proteomics: Emerging mass spectrometry-based approaches for single-cell protein analysis could reveal heterogeneity in HNRNPAB expression and modifications within tissues.

• Spatial transcriptomics combined with protein detection: These methods could correlate HNRNPAB protein localization with its RNA targets across tissue sections, providing spatial context to its functions.

• Nanobody-based detection: Development of nanobodies against HNRNPAB could enable super-resolution microscopy applications with improved spatial resolution compared to traditional antibodies.

These technologies may overcome current limitations in sensitivity, specificity, and resolution, potentially revealing new aspects of HNRNPAB biology in normal and pathological contexts.

How might understanding HNRNPAB function contribute to therapeutic developments?

The emerging roles of HNRNPAB in cancer progression suggest several promising therapeutic angles:

• Targeted inhibition: The correlation between HNRNPAB overexpression and poor prognosis in multiple cancers suggests it could be a valuable therapeutic target. Small molecule inhibitors or peptide mimetics disrupting its RNA-binding capacity could potentially suppress its oncogenic functions .

• Biomarker applications: HNRNPAB expression and subcellular localization patterns could serve as diagnostic or prognostic biomarkers. In hepatocellular carcinoma, cytoplasmic localization of HNRNPAB increases during dedifferentiation, suggesting utility as a risk assessment marker .

• Combination therapies: Understanding HNRNPAB's role in conferring treatment resistance could inform rational combination therapies. Its involvement in epithelial-mesenchymal transition suggests potential synergy with EMT-targeting approaches .

• RNA-based therapeutics: Since HNRNPAB regulates RNA processing, antisense oligonucleotides or RNA interference approaches targeting HNRNPAB-dependent splicing events might modulate disease progression.

• Precision medicine applications: The correlation between HNRNPAB expression and specific clinicopathological parameters suggests its potential use in patient stratification for personalized treatment approaches .

These therapeutic avenues highlight the translational potential of basic research into HNRNPAB biology, connecting fundamental RNA biology to clinical applications.

What are the most significant unanswered questions regarding HNRNPAB that future research should address?

Despite significant progress in HNRNPAB research, several critical questions remain:

• Mechanistic specificity: How does HNRNPAB, which shares significant homology with other hnRNP family members, achieve functional specificity in different cellular contexts? Comparative studies with related proteins like hnRNP A2/B1, E1/PCBP1, and K could reveal unique and shared functions .

• Regulatory networks: What upstream signals regulate HNRNPAB expression and localization during normal development and disease progression? The mechanisms governing its overexpression in cancers remain poorly understood .

• RNA targetome: What is the complete complement of RNAs regulated by HNRNPAB, and how does this change in disease states? Comprehensive RNA-immunoprecipitation studies coupled with next-generation sequencing could address this question.

• Structure-function relationships: How do specific domains of HNRNPAB contribute to its various functions, and which might serve as the most promising therapeutic targets?

• Systemic effects: Beyond cell-autonomous effects, how might HNRNPAB influence the tumor microenvironment or immune responses in cancer?

• Therapeutic resistance: Does HNRNPAB contribute to treatment resistance in cancers, and could its inhibition sensitize tumors to existing therapies?