HNRNPU Monoclonal Antibody

What is HNRNPU and why is it an important research target?

HNRNPU is a 825 amino acid protein that functions as a component of ribonucleosomes localized in cytoplasmic mRNP granules containing untranslated mRNAs . Its importance stems from its multifunctional nature, containing both DNA- and RNA-binding motifs, and its ability to shuttle between the nucleus and cytoplasm . Research has shown that HNRNPU regulates expression of several cytokines, including IL-6 and TNFα, by binding to the 3'-UTR of their mRNAs and stabilizing them . The protein's involvement in RNA processing and stability makes it a valuable target for understanding post-transcriptional regulation mechanisms.

What are the optimal applications for HNRNPU antibodies?

HNRNPU antibodies have been validated for multiple experimental applications, with different recommended dilutions for optimal results:

These applications have been tested and validated with human, mouse, and rat samples .

What is the correct molecular weight to expect when detecting HNRNPU?

While the calculated molecular weight of HNRNPU is 91 kDa based on its 825 amino acid sequence, the observed molecular weight on Western blots is typically 120 kDa . This discrepancy is likely due to post-translational modifications, particularly extensive phosphorylation. When running Western blots, researchers should look for bands at approximately 120 kDa rather than the calculated 91 kDa to properly identify HNRNPU.

What are the optimal buffer conditions for HNRNPU antibody storage and handling?

HNRNPU antibodies should be stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For long-term storage, maintain at -20°C where they remain stable for one year after shipment. Aliquoting is unnecessary for -20°C storage. For smaller sizes (20μl), the antibodies may contain 0.1% BSA. When handling these antibodies, avoid repeated freeze-thaw cycles to maintain antibody integrity and binding efficiency.

How should researchers optimize RNA-immunoprecipitation (RIP) assays for HNRNPU?

For RIP assays with HNRNPU, researchers should use cell lysates from appropriately stimulated cells (e.g., HeLa cells stimulated with P/I for 4 hours) . The anti-HNRNPU antibody for immunoprecipitation should be carefully selected; for example, Santa Cruz sc-32315 has been successfully used for IP in published research . Include normal mouse IgG as a negative control to assess non-specific binding. When validating RIP results, compare immunoprecipitated target RNA (e.g., IL-6) with housekeeping genes like RPLP0 to demonstrate specificity of binding . This methodology has successfully demonstrated that HNRNPU specifically binds IL-6 mRNA with minimal binding to housekeeping genes.

What is the recommended approach for subcellular fractionation when studying HNRNPU?

Since HNRNPU shuttles between the nucleus and cytoplasm, subcellular fractionation is crucial for understanding its compartment-specific functions. Research has shown that while HNRNPU is predominantly nuclear, it also has important cytoplasmic functions . When performing fractionation, use appropriate markers to validate separation: Lamin B1 for nuclear fractions and β-actin for cytoplasmic fractions . This is particularly important when investigating HNRNPU's role in mRNA stability, as interaction with target mRNAs like IL-6 has been specifically observed in the cytoplasmic fraction, suggesting compartment-specific functions .

How can CLIP-seq be optimized for studying HNRNPU-RNA interactions?

For comprehensive identification of HNRNPU RNA binding sites, an optimized crosslinking immunoprecipitation (CLIP) method is recommended. Research has shown that traditional HITS-CLIP protocols can be improved for HNRNPU by optimizing BrdU-CLIP methods . This modified approach produces approximately 10-fold greater yield of pre-amplified CLIP library, resulting in a low duplicate rate of CLIP-tag reads due to reduced PCR cycles required for library amplification . For HNRNPU CLIP experiments, consider:

• Initially using radioisotope labeling to confirm the position of RNA-RBP complexes on polyacrylamide gels

• Optimizing RNase concentration, lysate volume, and antibody conditions

• When working with subcellular fractions, adjusting protocols to accommodate smaller RNA amounts

This approach is particularly valuable for nucleocytoplasmic shuttling proteins like HNRNPU where compartment-specific interactions may vary significantly .

What controls should be included when studying HNRNPU's role in gene regulation?

When investigating HNRNPU's regulatory roles, several controls are essential:

• siRNA-mediated knockdown validation: Confirm knockdown efficiency by Western blot using validated antibodies (such as Bethyl A300-690A)

• Negative control siRNA: Include appropriate negative control siRNA (e.g., Silencer Select Negative Control)

• RIP assay controls: Include normal mouse IgG as negative control; compare binding to housekeeping genes (e.g., RPLP0) versus target genes to demonstrate specificity

• Subcellular localization controls: Use immunofluorescence with appropriate markers to validate HNRNPU localization patterns

Research has demonstrated that with proper controls, HNRNPU knockdown significantly decreases IL-6 mRNA in HeLa cells stimulated with P/I for 4 hours, and reduces IL-6 protein secretion after 24 hours of stimulation .

How can researchers resolve discrepancies between calculated and observed molecular weights of HNRNPU?

The discrepancy between calculated (91 kDa) and observed (120 kDa) molecular weights of HNRNPU presents a challenge for researchers . To resolve this:

• Always include positive controls from validated cell lines (HEK-293, HeLa, or Jurkat cells) where HNRNPU antibodies have been tested

• Consider the impact of post-translational modifications, particularly phosphorylation, which can significantly alter electrophoretic mobility

• When validating novel HNRNPU antibodies, perform knockdown or knockout experiments to confirm band specificity

• For definitive identification, consider mass spectrometry analysis of the immunoprecipitated protein

These approaches ensure accurate identification of HNRNPU despite the molecular weight discrepancy.

What are common pitfalls when performing immunofluorescence with HNRNPU antibodies?

When performing immunofluorescence with HNRNPU antibodies, researchers should be aware of several technical considerations:

• Dilution optimization: Different antibodies require different dilution ranges (14599-1-AP: 1:50-1:500; 16365-1-AP: 1:500-1:2000)

• Expected localization pattern: HNRNPU is predominantly nuclear but also has cytoplasmic localization; failure to detect cytoplasmic signal might indicate fixation or permeabilization issues

• Positive controls: Include validated cell lines such as HeLa cells for 14599-1-AP or HepG2 cells for 16365-1-AP

• Co-staining markers: Consider using nuclear markers (DAPI) and cytoplasmic markers (GAPDH has been used successfully) to validate subcellular localization patterns

Optimization of these parameters will help ensure specific and reproducible immunofluorescence staining with HNRNPU antibodies.

How can researchers address non-specific binding in HNRNPU immunoprecipitation experiments?

Non-specific binding in HNRNPU immunoprecipitation can be minimized through several approaches:

• Antibody selection: Choose antibodies validated for IP (Santa Cruz sc-32315 has been successfully used)

• Pre-clearing lysates: Incubate lysates with protein A/G beads before adding the specific antibody to reduce non-specific binding

• Blocking reagents: Include appropriate blocking reagents in washing buffers (such as BSA or non-fat milk)

• Stringent washing: Optimize wash buffers and washing steps without compromising specific interactions

• Appropriate controls: Always include isotype-matched normal IgG as negative control

These steps help ensure that observed interactions in IP experiments represent genuine HNRNPU-specific interactions rather than non-specific binding artifacts.

What factors affect HNRNPU detection in different experimental systems?

Several factors can influence successful detection of HNRNPU across experimental platforms:

• Cell/tissue type: While HNRNPU antibodies have been validated in human, mouse, and rat samples, expression levels and detection sensitivity may vary by tissue type

• Stimulation conditions: For studying HNRNPU's role in cytokine regulation, appropriate stimulation is critical (e.g., P/I stimulation for IL-6 regulation studies)

• Subcellular localization: HNRNPU's nucleocytoplasmic shuttling means that experimental conditions affecting this distribution can impact detection in specific compartments

• Post-translational modifications: Conditions affecting phosphorylation status may impact antibody recognition and apparent molecular weight

Understanding these variables allows researchers to optimize detection conditions for their specific experimental system.

How does HNRNPU regulate cytokine expression at the molecular level?

Research has revealed that HNRNPU regulates expression of several cytokines, including IL-6, by binding to their 3'-UTRs and stabilizing the mRNA . In HeLa cells stimulated with P/I, HNRNPU knockdown significantly decreased IL-6 mRNA levels and protein secretion . RIP assays confirmed that HNRNPU specifically binds IL-6 mRNA with minimal binding to housekeeping genes like RPLP0 . Importantly, this interaction was primarily observed in the cytoplasmic fraction, suggesting that cytoplasmic HNRNPU plays a crucial role in controlling mRNA stability . These findings align with earlier studies showing HNRNPU regulation of TNFα in 293T cells and IL-6/IL-1β in RAW264.7 macrophages stimulated with LPS .

What are the implications of HNRNPU's nucleocytoplasmic shuttling for experimental design?

HNRNPU's ability to shuttle between nuclear and cytoplasmic compartments has significant implications for experimental design:

• Compartment-specific functions: HNRNPU may have distinct roles in different cellular compartments; for example, it binds IL-6 mRNA primarily in the cytoplasm

• Fractionation approaches: When studying HNRNPU, consider subcellular fractionation to distinguish compartment-specific interactions

• CLIP considerations: For nucleocytoplasmic shuttling RBPs like HNRNPU, optimized CLIP methods that can work with smaller RNA amounts from specific subcellular fractions are particularly valuable

• Stimulus-dependent localization: Consider whether experimental stimuli affect HNRNPU's subcellular distribution

These considerations help design experiments that can accurately capture HNRNPU's multifaceted functions across different cellular compartments.

How can advanced techniques be adapted for studying HNRNPU in specialized cellular contexts?

For studying HNRNPU in specialized cellular contexts (such as specific organelles, neuronal dendrites, or axons), several adaptations are recommended:

• Optimized CLIP methods: The improved BrdU-CLIP method with 10-fold greater yield is particularly suitable for samples with limited RNA availability

• Reduced variance: Optimized protocols show reduced variance in yields and shortened experimental periods (by approximately 2 days)

• Subcellular fractionation approaches: Careful separation of cellular compartments with appropriate markers enables study of compartment-specific HNRNPU functions

• Integration of multiple techniques: Combining approaches (RIP, CLIP, subcellular fractionation, functional knockdown) provides comprehensive understanding of HNRNPU's role in specialized contexts

These adaptations enable researchers to investigate HNRNPU's functions in specific cellular microenvironments with greater precision and efficiency.