HNRNPK Antibody

What is HNRNPK and why is it significant for research?

HNRNPK belongs to the heterogeneous nuclear ribonucleoprotein family comprising approximately 20 proteins that participate in a wide range of key cellular functions. These proteins are involved in numerous pathways implicated, disrupted, or dysregulated in tumor development and progression. HNRNPK functions as a conserved pre-mRNA-binding protein with critical roles in RNA processing and maintenance .

The significance of HNRNPK in research stems from its multifunctional nature, playing important roles in cancer cell proliferation and metastasis . Recent studies have revealed its novel role as an IRES-transacting factor (ITAF) that stimulates IRES-mediated translation initiation for retroviruses including HIV-1 and HTLV-1 . Its ability to maintain RNAs in single-stranded form by preventing RNA-RNA interactions has fundamental implications for gene expression regulation .

What applications are HNRNPK antibodies validated for?

HNRNPK antibodies have been validated for multiple research applications as demonstrated in the table below:

It is recommended that researchers titrate these antibodies in their specific testing systems to obtain optimal results, as dilution requirements may be sample-dependent .

What species reactivity do commonly available HNRNPK antibodies demonstrate?

Most commercially available HNRNPK antibodies show broad cross-reactivity across multiple species. Based on the search results, HNRNPK antibody reactivity has been confirmed in:

• Human

• Mouse

• Rat

• Cow

• Xenopus laevis

• Hamster

Some antibodies may also react with samples from rabbit, chicken, dog, zebrafish, guinea pig, goat, horse, monkey, pig, and bat, though reactivity varies by specific antibody clone and manufacturer .

What is the correct molecular weight for HNRNPK detection in Western blot applications?

When performing Western blot analysis for HNRNPK, researchers should expect to observe a band at approximately 60 kDa, which represents the observed molecular weight. This differs slightly from the calculated molecular weight of 51 kDa (463 amino acids) . This discrepancy is common for many proteins due to post-translational modifications and structural characteristics that affect migration during SDS-PAGE. Verifying this correct molecular weight is essential for experimental validation and avoiding false positive or negative results.

How can HNRNPK antibodies be utilized to study its role in viral RNA translation?

Recent research has uncovered HNRNPK's function as an IRES-transacting factor (ITAF) that promotes cap-independent translation initiation for retroviral mRNAs. When investigating this function, researchers should:

• Design experiments that compare cap-dependent and IRES-mediated translation in the presence and absence of HNRNPK

• Utilize HNRNPK antibodies for immunoprecipitation (IP) followed by RNA isolation to identify viral RNA-protein interactions

• Perform Western blotting after manipulating HNRNPK levels (depletion or overexpression) to assess changes in viral protein synthesis

• Combine immunofluorescence with RNA FISH techniques to visualize co-localization of HNRNPK with viral RNA

Studies have demonstrated that in HIV-1-expressing cells, the depletion of HNRNPK reduced HIV-1 vRNA translation, and both depletion and overexpression of HNRNPK modulated HIV-1 IRES activity. Additionally, HNRNPK has been shown to act as an ITAF for the human T cell lymphotropic virus-type 1 (HTLV-1) IRES present in the 5′UTR of the viral sense mRNA .

What considerations are important when studying HNRNPK post-translational modifications?

When investigating post-translational modifications (PTMs) of HNRNPK, researchers should consider:

• Phosphorylation and asymmetrical dimethylation (aDMA) of HNRNPK significantly impact its function in cap-independent translation

• Protein arginine methyltransferase 1 (PRMT1)-induced asymmetrical dimethylation specifically affects HNRNPK's ability to promote HIV-1 IRES activity

• Phosphorylation at Ser284 can be detected using phospho-specific antibodies

Methodology recommendations:

• Use phospho-specific antibodies (e.g., anti-HNRNPK pSer284) for detecting specific phosphorylation events

• Employ two-dimensional gel electrophoresis before Western blotting to separate differently modified forms

• Consider using λ-phosphatase treatment as a control to confirm phosphorylation status

• Use IP with anti-HNRNPK antibodies followed by Western blotting with modification-specific antibodies (anti-methyl, anti-phospho)

These approaches help elucidate how post-translational modifications regulate HNRNPK's diverse functions in RNA processing and viral translation .

What methodological approaches should be used to investigate HNRNPK's role in RNA-RNA interactions?

HNRNPK has been shown to play a critical role in maintaining RNAs in single-stranded form by preventing RNA-RNA interactions. When investigating this function:

• Design RNA immunoprecipitation (RIP) experiments using HNRNPK antibodies to identify bound RNA species

• Perform HnRNPK loss-of-function and gain-of-function experiments to assess changes in global single- and double-stranded RNA levels

• Analyze subcellular localization changes of target RNAs following HNRNPK depletion

• Use crosslinking and immunoprecipitation (CLIP) methods with HNRNPK antibodies to identify direct RNA binding sites

Research has shown that HNRNPK depletion can neutralize the oncogenic functions of certain RNAs by promoting double-stranded RNA formation and cytoplasmic accumulation. For example, with the sense-antisense pair IER3 and IER3-AS1, HNRNPK controls both mRNA stability and colocalization, with its interaction determining their oncogenic functions by maintaining them in single-stranded form .

How can researchers optimize HNRNPK antibody-based immunohistochemistry for cancer tissue analysis?

When optimizing immunohistochemistry (IHC) protocols for HNRNPK detection in cancer tissues:

• Antigen retrieval method selection is critical:

  • Primary recommendation: Use TE buffer at pH 9.0

  • Alternative approach: Use citrate buffer at pH 6.0 if optimal results aren't achieved with TE buffer

• Dilution optimization:

  • Start with the recommended range of 1:500-1:2000

  • Perform a dilution series to identify optimal signal-to-noise ratio for specific tissue types

• Positive control selection:

  • Human colon cancer tissue

  • Human breast cancer tissue

• Consider antibody isotype:

  • For mouse monoclonal antibodies of IgA isotype, use "anti-mouse IgG (H+L)" secondary antibodies

  • For IgM or IgG2b isotypes, ensure appropriate secondary antibody selection

This optimization is essential as HNRNPK expression has been implicated in cancer cell proliferation and metastasis, making it a potential biomarker for cancer research .

What storage and handling practices ensure optimal HNRNPK antibody performance?

To maintain HNRNPK antibody integrity and performance:

• Storage conditions:

  • Store at -20°C

  • Antibodies formulated with 50% glycerol remain stable for one year after shipment

  • Aliquoting is generally unnecessary for -20°C storage with glycerol-containing formulations

• Buffer composition considerations:

  • Most HNRNPK antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Some preparations (20μl sizes) may contain 0.1% BSA as a stabilizer

• Freeze-thaw cycle management:

  • While glycerol formulations are resistant to freeze-thaw damage, minimize repeated cycles

  • Return antibodies to -20°C promptly after use

• Working dilution preparation:

  • Prepare fresh working dilutions on the day of experiment

  • Dilute in appropriate buffers containing 0.1-0.5% BSA as a stabilizer

Proper storage and handling significantly impact experimental reproducibility and antibody longevity .

How can researchers verify HNRNPK antibody specificity for their experimental system?

To ensure antibody specificity:

• Positive controls:

  • For Western blot: Use lysates from validated cell lines including LNCaP, HuH-7, HeLa, HEK-293, Jurkat, K-562, HSC-T6, PC-12, NIH/3T3, or RAW 264.7 cells

  • For IHC: Use human colon cancer tissue or human breast cancer tissue

• Negative controls:

  • Include HNRNPK knockdown/knockout samples

  • Use isotype control antibodies at equivalent concentrations

  • Include blocking peptide competition assays when available

• Band verification:

  • Confirm the 60 kDa band corresponds to HNRNPK (vs. calculated 51 kDa)

  • Be aware that post-translational modifications may alter migration patterns

• Cross-reactivity assessment:

  • When using antibodies across species, perform validation in each species

  • Verify epitope conservation through sequence alignment

What are the potential pitfalls in interpreting HNRNPK antibody results in RNA-binding protein studies?

When interpreting HNRNPK antibody results:

• Functional redundancy considerations:

  • HNRNPK belongs to a family of 20 heterogeneous nuclear ribonucleoproteins

  • Other family members may compensate for HNRNPK in knockdown experiments

• Context-dependent function interpretation:

  • HNRNPK may exhibit different functions depending on cellular context

  • Post-translational modifications significantly alter function but may not affect antibody recognition

• Subcellular localization challenges:

  • HNRNPK shuttles between nucleus and cytoplasm

  • Fixation methods may affect localization patterns in IF/ICC experiments

• RNA-protein complex integrity:

  • Standard fixation methods may disrupt RNA-protein interactions

  • Consider using cross-linking approaches to preserve native complexes

• Differential splice variant detection:

  • Ensure the antibody epitope is present in all relevant splice variants

  • Verify splice variant expression in your experimental system

These considerations help avoid misinterpretation of results, particularly when studying HNRNPK's complex roles in RNA-protein interactions .

How can HNRNPK antibodies be applied to study cancer mechanisms?

HNRNPK has been implicated in cancer cell proliferation and metastasis, making it an important target for cancer research . When investigating HNRNPK in cancer models:

• Expression analysis approaches:

  • Perform IHC on cancer tissue microarrays to correlate expression with clinical outcomes

  • Use Western blotting to compare expression levels across cancer cell lines and normal counterparts

  • Combine with subcellular fractionation to detect compartment-specific alterations

• Functional analysis strategies:

  • Use ChIP with HNRNPK antibodies to identify cancer-specific DNA binding sites

  • Perform RIP to identify cancer-relevant RNA targets

  • Combine with proximity ligation assays to detect cancer-specific protein interaction partners

• Mechanistic studies:

  • Investigate HNRNPK's role in maintaining oncogenic single-stranded RNAs

  • Study how HNRNPK depletion affects double-stranded RNA formation and cytoplasmic accumulation in cancer cells

  • Explore the relationship between HNRNPK and FGF-2 regulated transcriptome in normal versus cancer cells

These approaches can reveal how HNRNPK contributes to cancer progression through its diverse molecular functions .

What considerations are important when applying HNRNPK antibodies in virus infection studies?

When studying viral infections using HNRNPK antibodies:

• Temporal dynamics considerations:

  • Monitor HNRNPK expression, localization, and modification changes throughout the viral life cycle

  • Compare early vs. late infection timepoints to capture dynamic changes

• Viral translation specific approaches:

  • Use bicistronic reporter constructs containing viral IRES elements to study HNRNPK's ITAF activity

  • Combine HNRNPK antibodies with viral protein detection to correlate HNRNPK activity with viral translation

• IRES-specific methodologies:

  • Compare HNRNPK's role across different viral IRES elements (HIV-1 vs. HTLV-1)

  • Investigate how HNRNPK phosphorylation and methylation impact IRES-mediated translation

  • Study differential effects on viral sense vs. antisense transcripts (e.g., HTLV-1 vs. sHBZ)

• Host-pathogen interaction analysis:

  • Investigate how viral proteins might modify HNRNPK function through direct interaction

  • Examine changes in HNRNPK post-translational modifications during infection

These approaches help elucidate HNRNPK's role in promoting cap-independent translation of retroviral mRNAs, which is crucial for understanding viral pathogenesis .

What emerging research directions might benefit from HNRNPK antibody applications?

Several cutting-edge research areas could benefit from HNRNPK antibody applications:

• RNA therapeutics development:

  • Understanding HNRNPK's role in maintaining RNA single-strandedness could inform antisense oligonucleotide design

  • HNRNPK antibodies could help screen for compounds that modulate RNA-RNA interactions

• Liquid-liquid phase separation (LLPS) biology:

  • HNRNPK's role in ribonucleoprotein complexes makes it relevant to biomolecular condensate research

  • Antibodies could help characterize HNRNPK's participation in stress granules and processing bodies

• Epitranscriptomics:

  • HNRNPK likely interacts with modified RNAs

  • Antibodies could help identify how RNA modifications affect HNRNPK binding

• Single-cell analysis technologies:

  • Adapting HNRNPK antibodies for single-cell protein analysis

  • Combining with single-cell transcriptomics to correlate HNRNPK levels with gene expression patterns

• CRISPR screening approaches:

  • Using HNRNPK antibodies to validate hits from CRISPR screens targeting RNA processing pathways

  • Developing HNRNPK protein reporters for live-cell CRISPR screening