HNRNPH1 Antibody

What is HNRNPH1 and what are its basic cellular functions?

HNRNPH1 (heterogeneous nuclear ribonucleoprotein H1) is an RNA-binding protein that plays crucial roles in pre-mRNA processing events. It is part of the hnRNP complex that provides the substrate for processing events that pre-mRNAs undergo before maturation. HNRNPH1 has been identified as a protein with significant roles in viral defense mechanisms and oncological contexts. It has a calculated molecular weight of 49 kDa, though it is typically observed at 49-50 kDa in experimental contexts . The protein is involved in various cellular processes including RNA splicing, nucleocytoplasmic transport, and regulation of gene expression. Recent research has identified HNRNPH1 as an important regulator of cellular proliferation in certain cancer contexts .

What applications can HNRNPH1 antibody be used for in research?

HNRNPH1 antibody has been validated for multiple experimental applications, allowing researchers to investigate this protein in various contexts. Based on published literature and validation data, the antibody can be reliably used in the following applications:

These applications have been confirmed with multiple cell and tissue types, including HEK-293T cells, mouse embryonic stem cells, HeLa cells, HepG2 cells, and brain tissue from mouse and rat models . It's recommended that researchers optimize dilutions for their specific experimental systems to obtain optimal results.

How should researchers properly store and handle HNRNPH1 antibody?

Proper storage and handling of HNRNPH1 antibody is critical for maintaining its efficacy and obtaining reliable experimental results. The antibody should be stored at -20°C where it remains stable for one year after shipment. The storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is unnecessary for -20°C storage, which simplifies laboratory handling procedures. For the 20μl size format, it's worth noting that the preparation contains 0.1% BSA .

When working with the antibody, researchers should avoid repeated freeze-thaw cycles, maintain sterile handling conditions, and keep the antibody on ice during experiments to preserve its binding capacity. Before use, the antibody should be gently mixed rather than vortexed to prevent protein denaturation that could affect binding specificity.

How can researchers verify the specificity of HNRNPH1 antibody in their experimental systems?

Verifying antibody specificity is essential for generating reliable research data. For HNRNPH1 antibody, researchers should implement a multi-faceted validation approach:

First, perform Western blot analysis with positive controls known to express HNRNPH1, such as HEK-293T, HeLa, or HepG2 cells, which should yield a band at approximately 49-50 kDa . The inclusion of a negative control is equally important—either using cells where HNRNPH1 has been knocked down via siRNA or using tissues/cells known not to express the protein.

Second, conduct peptide competition assays by pre-incubating the antibody with excess immunizing peptide before application in your experimental protocol. This should abolish or significantly reduce specific binding if the antibody is truly targeting HNRNPH1.

Third, replicate your findings with a second HNRNPH1 antibody from a different source or one raised against a different epitope of the protein. Concordant results would strongly support specificity.

Fourth, for definitive validation, perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is truly capturing HNRNPH1 protein in your experimental system.

In RNA-related studies, researchers can verify specificity through RNA immunoprecipitation (RIP) assays, as demonstrated in recent studies where biotinylated RNA oligomers were used to pull down HNRNPH1, followed by Western blot analysis using the anti-HNRNPH1 antibody .

What are the methodological considerations for studying HNRNPH1's role in viral infection using the antibody?

When investigating HNRNPH1's role in viral infections, particularly influenza A virus (IAV), several methodological considerations are essential:

First, experimental design should incorporate both gain-of-function (overexpression) and loss-of-function (knockdown) approaches to comprehensively evaluate HNRNPH1's impact. Research has demonstrated that overexpression of HNRNPH1 in A549 cells significantly decreases IAV multiplication in a dose-dependent manner, while knockdown enhances viral replication .

For protein interaction studies, co-immunoprecipitation (Co-IP) assays have proven effective in demonstrating HNRNPH1's interaction with viral proteins like NS1. When performing these experiments, researchers should be aware that the interaction between HNRNPH1 and NS1 is RNA-dependent, as demonstrated by surface plasmon resonance (SPR) assays with a KD value of approximately 10.37 μM .

Temporal considerations are critical—growth curve analysis shows that HNRNPH1's effect on viral multiplication varies at different time points post-infection (12, 24, 36 h.p.i.). Both plaque assays and HA titer assays should be employed to quantify viral titers .

For subcellular localization studies, immunofluorescence assays reveal that HNRNPH1 undergoes nucleocytoplasmic shuttling upon IAV infection, with increasing cytoplasmic localization observed from 6 to 8 hours post-infection. This dynamic relocalization correlates with HNRNPH1's functional impact on viral replication .

When designing experiments to test HNRNPH1's broad-spectrum antiviral activity, include multiple IAV subtypes. Current evidence suggests that HNRNPH1 overexpression inhibits both H3N2 (A/Aichi/2/1968) and WSN (A/WSN/1933 (H1N1)) subtypes, though with variable efficacy .

How should researchers quantify and interpret HNRNPH1 expression changes in oncological contexts?

In oncological research contexts, accurate quantification and interpretation of HNRNPH1 expression changes require rigorous methodological approaches:

For baseline expression analysis, researchers should employ multiple cell lines relevant to the cancer type being studied, alongside appropriate non-malignant controls. In chronic myeloid leukemia (CML) research, for example, HNRNPH1 has been found to be upregulated in both patient samples and cell lines .

When quantifying HNRNPH1 at the protein level, Western blot analysis should be complemented with densitometry for semi-quantitative assessment. For mRNA quantification, qRT-PCR remains the gold standard, with careful selection of reference genes appropriate for the cell types being compared.

Functional studies should incorporate colony formation assays, which have proven effective in evaluating the impact of HNRNPH1 modulation on cellular proliferation. These assays should follow standard procedures using 1% methylcellulose medium supplemented with 40% FBS and 1% penicillin/streptomycin, with colonies counted after 14-18 days of incubation. Colonies should be defined as aggregates containing more than 50 cells as observed under a microscope .

For mechanistic investigations, RNA immunoprecipitation can be performed using anti-HNRNPH1 antibody (such as Abcam No 154894) to identify direct RNA targets. This approach should include appropriate controls, such as IgG immunoprecipitation and input RNA samples .

Transcriptome-wide effects of HNRNPH1 modulation should be assessed using microarray or RNA-seq analysis, comparing control cells with those where HNRNPH1 has been knocked down. For reliable results, at least three biological replicates should be analyzed for each experimental condition .

What considerations should be taken when using HNRNPH1 antibody for studying its interactions with RNA structures?

When investigating HNRNPH1's interactions with RNA structures, researchers should consider several critical methodological aspects:

HNRNPH1 demonstrates specific binding to certain RNA motifs and structures, particularly G-quadruplexes and G-rich sequences. When designing RNA binding experiments, include both wild-type and mutant RNA oligomers as controls. For instance, in studies of fusion oncogene regulation, rG1 RNA oligomers and control rG1 mt1 variants have been used to demonstrate specificity .

For in vitro binding assays, electrophoretic mobility shift assays (EMSA) with chemiluminescent detection have proven effective for assessing HNRNPH1:RNA interactions. When performing these assays, titrate purified HNRNPH1 protein to establish concentration-dependent binding relationships .

Competition assays provide valuable validation of binding specificity. These can be performed using antibody-based RNA binding assays with constant concentrations of purified HNRNPH1 and biotinylated RNA, while varying concentrations of non-biotinylated RNA serve as competitors. Previous research has established an IC50 of 84 nM for rG1 RNA competition .

For cellular validation of RNA interactions, transfect cells with biotinylated RNA oligomers at varying concentrations (e.g., 24-hour transfection), followed by UV crosslinking, streptavidin pull-down, and Western blot analysis using anti-HNRNPH1 antibody. This approach has successfully demonstrated concentration-dependent enrichment of HNRNPH1 binding to specific RNA sequences in cellular contexts .

When evaluating the functional impact of HNRNPH1-RNA interactions, measure both mRNA and protein levels of potential targets. For example, qPCR analysis has shown that rG1 RNA oligomers can reduce EWS-FLI1 mRNA levels by approximately 50% at concentrations of 80-160 nM, with corresponding reductions in protein levels observed at concentrations ≥40 nM .

What are the optimal conditions for immunohistochemistry using HNRNPH1 antibody?

For successful immunohistochemistry (IHC) using HNRNPH1 antibody, researchers should adhere to specific technical conditions for optimal results:

Antigen retrieval is a critical step that significantly impacts staining quality. For HNRNPH1 antibody, TE buffer at pH 9.0 is the suggested primary method for antigen retrieval. Alternatively, researchers may use citrate buffer at pH 6.0, though comparative studies between these methods have not been comprehensively published . This choice of buffer may be particularly important when working with formalin-fixed, paraffin-embedded tissues where protein cross-linking can mask epitopes.

Validation data indicates that HNRNPH1 antibody has been successfully used for IHC in human cervical cancer tissue . When extending this application to other tissue types, appropriate positive and negative controls should be included. For negative controls, either omit the primary antibody or use isotype-matched control immunoglobulin.

Signal development systems should be selected based on the type of microscopy to be performed. For brightfield microscopy, DAB (3,3'-diaminobenzidine) is commonly used, while fluorescent secondary antibodies are appropriate for fluorescence microscopy.

How can researchers optimize Western blot protocols for HNRNPH1 detection in different cell types?

Optimizing Western blot protocols for HNRNPH1 detection requires careful consideration of several technical parameters:

Sample preparation is critical—when lysing cells, use buffer containing appropriate protease inhibitors to prevent degradation of HNRNPH1. The protein has been successfully detected in various cell types including HEK-293T, mouse embryonic stem cells, HeLa, HepG2, and brain tissue from both mouse and rat .

For protein separation, standard SDS-PAGE using 10-12% polyacrylamide gels is generally sufficient to resolve HNRNPH1, which has an observed molecular weight of 49-50 kDa . Transfer conditions should be optimized for proteins in this molecular weight range—typically, a semi-dry transfer system using PVDF membrane with 25V for 30 minutes or wet transfer at 100V for 60-90 minutes.

Blocking conditions can significantly impact antibody specificity. A 5% non-fat dry milk or BSA solution in TBS-T (Tris-buffered saline with 0.1% Tween-20) for 1-2 hours at room temperature is typically effective for reducing non-specific binding.

The recommended dilution range for HNRNPH1 antibody in Western blot applications is 1:1000-1:6000 . For optimal results, researchers should first perform a dilution series within this range using a sample known to express HNRNPH1. Incubation with primary antibody is generally performed overnight at 4°C with gentle agitation to ensure even antibody distribution.

For detection, both chemiluminescence and fluorescence-based secondary antibody systems are compatible. When quantitative analysis is required, fluorescence-based systems offer advantages in terms of dynamic range and multiplexing capability.

What controls should be included when performing co-immunoprecipitation with HNRNPH1 antibody?

When performing co-immunoprecipitation (Co-IP) with HNRNPH1 antibody to investigate protein-protein interactions, several critical controls should be included to ensure result validity:

An input control is essential, representing a small portion (typically 5-10%) of the whole cell lysate before immunoprecipitation. This allows for comparison of the relative abundance of proteins in the starting material versus the immunoprecipitated fraction. In published protocols, researchers have typically reserved 10% of the lysate as input control .

A negative control immunoprecipitation using isotype-matched non-specific IgG (such as rabbit IgG for rabbit-derived HNRNPH1 antibody) is crucial to identify non-specific binding. This control should undergo identical experimental conditions as the HNRNPH1 antibody immunoprecipitation. Research protocols typically allocate 10% of lysate for this control .

For suspected protein interactions, reciprocal Co-IP provides powerful validation. If HNRNPH1 is found to interact with Protein X, perform a second Co-IP using antibodies against Protein X and probe for HNRNPH1. This approach has been successfully employed to confirm interaction between HNRNPH1 and NS1 protein, where both HNRNPH1-Flag could co-immunoprecipitate NS1-Myc and vice versa .

RNA-dependency controls should be included when studying RNA-binding proteins like HNRNPH1. Treat duplicate samples with RNase before immunoprecipitation to determine if the observed interactions are direct protein-protein interactions or mediated by RNA. Previous research has demonstrated that HNRNPH1's interaction with viral proteins can be RNA-dependent .

Competitive peptide controls, where the immunoprecipitation is performed in the presence of excess peptide used to generate the antibody, can establish the specificity of the antibody-mediated pull-down.

What are common issues in immunofluorescence studies with HNRNPH1 antibody and how can they be resolved?

Researchers frequently encounter several challenges when performing immunofluorescence studies with HNRNPH1 antibody, but these can be systematically addressed:

High background fluorescence is a common issue that can obscure specific HNRNPH1 signals. This problem can be mitigated by optimizing blocking conditions (try 3-5% BSA or normal serum matching the species of the secondary antibody), extending blocking time to 1-2 hours at room temperature, and increasing the number or duration of washing steps with PBS containing 0.1-0.3% Triton X-100. Additionally, verify that secondary antibody concentrations are not excessive—typically a 1:500-1:1000 dilution is appropriate.

Poor or weak signal detection despite known HNRNPH1 expression can result from insufficient antigen retrieval or suboptimal antibody concentration. For fixed cells, test different permeabilization methods (0.1-0.5% Triton X-100 for 5-15 minutes) and ensure the primary antibody concentration falls within the recommended range of 1:50-1:500 for immunofluorescence applications . Extending primary antibody incubation to overnight at 4°C may enhance signal detection.

Inconsistent subcellular localization patterns between experiments might reflect technical variability or biological changes in HNRNPH1 distribution. HNRNPH1 is predominantly nuclear but can shuttle between nucleus and cytoplasm, especially during viral infection . Standardize cell fixation timing and methods, as variations in cellular stress can affect HNRNPH1 localization. When studying dynamic processes like viral infection, carefully document and maintain consistent time points post-treatment (e.g., 6-8 hours post-infection has shown significant HNRNPH1 relocalization) .

Non-specific nuclear staining can be distinguished from specific HNRNPH1 signals by including HNRNPH1-knockdown cells as negative controls. Additionally, compare staining patterns with published results showing positive HNRNPH1 detection in HepG2 cells .

How can researchers troubleshoot false positive or false negative results in HNRNPH1 detection?

Addressing false positive or false negative results in HNRNPH1 detection requires systematic troubleshooting approaches:

For false positives in Western blot or immunofluorescence, first verify antibody specificity through multiple controls. Include HNRNPH1 knockdown samples created using siRNA (such as sihnRNPH1-1, which has shown superior knockdown efficiency compared to sihnRNPH1-2) . If using overexpression systems, employ epitope-tagged constructs (like hnRNPH1-Flag) to differentiate between endogenous and exogenous protein using tag-specific antibodies .

Cross-reactivity with related proteins, particularly other hnRNP family members with similar molecular weights, can cause false positive results. To address this, utilize more stringent washing conditions and higher dilutions of primary antibody. Additionally, compare detection patterns with antibodies targeting related proteins to identify potential cross-reactivity.

False negatives often result from technical issues with protein extraction or detection. HNRNPH1's nuclear localization requires efficient nuclear protein extraction methods. Use nuclear extraction protocols with NP-40 or RIPA buffer containing appropriate protease inhibitors. For fixed tissue samples, optimize antigen retrieval methods—while TE buffer at pH 9.0 is recommended, some applications may require citrate buffer at pH 6.0 .

Sample handling can significantly impact results—HNRNPH1 is relatively stable but improper storage or repeated freeze-thaw cycles of samples can lead to protein degradation. Prepare fresh lysates when possible and add protease inhibitors immediately after cell lysis.

Interference from post-translational modifications may affect epitope recognition. If HNRNPH1 is suspected to undergo modifications in your experimental system (such as during viral infection), consider using multiple antibodies targeting different epitopes of the protein to ensure detection regardless of modification status.

What methodological modifications are needed when studying HNRNPH1 across different species models?

When investigating HNRNPH1 across different species models, researchers must adapt their methodological approaches to account for interspecies variations:

Dilution optimization should be performed independently for each species model. While the recommended dilution ranges (1:1000-1:6000 for WB, 1:50-1:500 for IHC/IF) provide starting points, species-specific optimal concentrations may vary. Perform dilution series experiments with positive control samples from each species to determine optimal working concentrations.

For RNA interaction studies across species, account for potential differences in RNA binding preferences. The RNA dependency of HNRNPH1's interactions with other proteins, such as viral NS1, suggests that species-specific RNA structure variations could impact experimental outcomes . When designing RNA oligonucleotides for binding studies, consider incorporating species-specific sequence variations.

In functional assays measuring HNRNPH1's impact on viral replication or gene expression, species-specific cell lines should be employed to account for potential differences in cellular pathways. For example, when studying HNRNPH1's effect on influenza virus replication, both human (A549) and relevant animal cell lines should be tested if cross-species comparisons are intended .

For immunofluorescence studies, fixation and permeabilization protocols may require species-specific optimization. Cell types from different species may respond differently to standard fixatives (paraformaldehyde, methanol) and permeabilization agents (Triton X-100, saponin). Test multiple conditions to determine optimal parameters for each species-derived sample.

How might HNRNPH1 antibody be used to explore potential therapeutic applications in viral infections?

HNRNPH1 antibody offers valuable research tools for investigating potential therapeutic strategies targeting viral infections, particularly influenza:

Mechanistic studies can utilize HNRNPH1 antibody to elucidate the protein's antiviral pathways. Since HNRNPH1 overexpression has been shown to decrease influenza A virus (IAV) multiplication while its knockdown enhances viral replication , researchers should employ immunoprecipitation followed by mass spectrometry to identify the complete interactome of HNRNPH1 during viral infection. This approach could reveal additional host factors involved in HNRNPH1-mediated antiviral responses.

Domain-mapping experiments using truncated HNRNPH1 constructs and co-immunoprecipitation with HNRNPH1 antibody can pinpoint specific regions responsible for interaction with viral proteins like NS1. Previous research has indicated that the RBD domain of NS1 and the RRM and NLS regions of HNRNPH1 may be critical interaction sites . Detailed mapping could inform the design of peptide inhibitors that disrupt virus-host protein interactions.

High-throughput screening approaches could identify small molecules that enhance HNRNPH1's antiviral activity. Following compound treatment, researchers can quantify HNRNPH1 expression and localization using the antibody in immunofluorescence or Western blot assays. Promising compounds would be those that upregulate HNRNPH1 expression or promote its interaction with viral factors in ways that inhibit viral replication.

For translational research, animal models of influenza infection could be utilized to assess whether modulating HNRNPH1 expression affects disease progression and viral clearance. HNRNPH1 antibody would be essential for confirming successful genetic manipulation or therapeutic intervention in these models.

Cross-viral studies should investigate whether HNRNPH1's antiviral properties extend beyond influenza to other RNA viruses. Since HNRNPH1 has demonstrated inhibitory effects on different IAV subtypes (H3N2 and H1N1) , broader antiviral potential is possible and could be explored using the antibody to track HNRNPH1 dynamics during infection with various viral pathogens.

What emerging techniques could enhance the utility of HNRNPH1 antibody in cancer research?

Emerging technologies present exciting opportunities to expand HNRNPH1 antibody applications in cancer research contexts:

Single-cell proteomics approaches could leverage HNRNPH1 antibody to examine expression heterogeneity within tumors. Given HNRNPH1's upregulation in certain cancer contexts like chronic myeloid leukemia , mass cytometry (CyTOF) or imaging mass cytometry using metal-conjugated HNRNPH1 antibody could map expression patterns at single-cell resolution across tumor microenvironments, potentially identifying subpopulations with distinct HNRNPH1 expression profiles.

Proximity ligation assays (PLA) using HNRNPH1 antibody paired with antibodies against suspected interaction partners could visualize and quantify protein-protein interactions in situ within cancer tissues. This technique offers advantages over traditional co-immunoprecipitation by preserving spatial information and detecting transient interactions.

CRISPR screening combined with HNRNPH1 antibody-based detection could identify genetic dependencies related to HNRNPH1 function in cancer cells. After CRISPR library infection, high-content imaging using fluorescently-labeled HNRNPH1 antibody could identify genes whose knockout alters HNRNPH1 expression, localization, or interaction patterns.

Spatial transcriptomics paired with HNRNPH1 immunofluorescence could correlate HNRNPH1 protein expression with local transcriptional programs in tumor sections. This integrated approach would provide insights into how HNRNPH1's RNA processing functions shape the transcriptional landscape within specific tumor regions.

Liquid biopsy applications might utilize HNRNPH1 antibody to detect cancer-derived extracellular vesicles (EVs). If HNRNPH1 is packaged into cancer-derived EVs, immunocapture using the antibody followed by molecular profiling could provide minimally invasive biomarkers for cancers where HNRNPH1 plays a significant role.

Patient-derived organoid models could be characterized for HNRNPH1 expression using the antibody, then subjected to functional studies through genetic manipulation or drug treatment. This approach would bridge the gap between cell line research and clinical applications, potentially identifying patient subgroups likely to benefit from therapies targeting HNRNPH1-dependent pathways.