HNRNPR Antibody

What is HNRNPR and what are its main biological functions?

HNRNPR is a heterogeneous nuclear ribonucleoprotein that acts as a component of ribonucleosomes, which are complexes containing at least 20 different hnRNP proteins. It plays crucial roles in multiple RNA processing pathways, specifically:

• Pre-mRNA processing in the nucleus

• mRNA splicing as part of the spliceosome C complex

• RNA transport between cellular compartments

• Regulation of mRNA stability

• Promotion of transcription at specific genes such as c-fos

HNRNPR has a modular structure featuring three adjacent RNA binding domains in its central region, followed by a glycine- and arginine-rich section (RGG box) in the C-terminal portion that forms another type of RNA binding motif. This structural arrangement enables its multifaceted roles in RNA metabolism .

What is the molecular structure and organization of HNRNPR protein?

HNRNPR exhibits a defined modular structure critical for its function:

• An acidic N-terminal region of approximately 150 amino acids

• Three adjacent consensus sequence RNA binding domains located in the central part

• A nuclear localization signal in the C-terminal portion

• An octapeptide (PPPRMPPP) with similarity to a major B cell epitope of the snRNP core protein B

• A glycine- and arginine-rich section of approximately 120 amino acids forming an RGG box

• Three copies of a tyrosine-rich decapeptide interspersed in the RGG box region

The full-length protein has a calculated molecular weight of 71kDa, though it often appears at around 78-82kDa in Western blot analyses due to post-translational modifications .

How does HNRNPR differ from other heterogeneous nuclear ribonucleoproteins?

While HNRNPR shares functional similarities with other hnRNP family members, it possesses distinctive features:

• HNRNPR shows immunological relationship with hnRNP P

• It contains a unique combination of three adjacent RNA binding domains plus an RGG box

• Unlike some other hnRNPs, it specifically binds to 7SK noncoding RNA through stem-loop structures

• It has distinctive tyrosine-rich decapeptide repeats in its RGG box region

• HNRNPR generates two isoforms through alternative splicing of exon 2, which differ at the N-terminus

This unique structural arrangement contributes to HNRNPR's specialized functions in RNA processing pathways.

What are the validated applications for HNRNPR antibodies in research?

HNRNPR antibodies have been validated for multiple experimental applications:

Researchers should validate each application with appropriate positive and negative controls for their specific experimental system .

How should researchers validate HNRNPR antibody specificity for their experiments?

Thorough validation of HNRNPR antibody specificity is critical for reliable experimental outcomes:

• Knockout validation: Use HNRNPR knockout cell lines (e.g., HNRNPR knockout HEK-293T) as negative controls to confirm absence of signal

• Multiple antibody comparison: Compare results using antibodies targeting different epitopes of HNRNPR (e.g., antibodies specific for C-terminus versus N-terminus)

• Molecular weight verification: Confirm detection at the expected molecular weight (~71-82 kDa)

• Immunodepletion: Pre-absorb antibody with recombinant HNRNPR protein to demonstrate signal reduction

• Cellular localization pattern: Verify expected nuclear and nucleoplasmic localization pattern

• Cross-reactivity assessment: Test antibody against related hnRNP family members to ensure specificity

A comprehensive validation approach using multiple methods provides confidence in antibody specificity and experimental reliability.

What are the optimal protocols for Western blot detection of HNRNPR?

For optimal Western blot detection of HNRNPR, researchers should follow these methodological recommendations:

• Sample preparation:

  • Use RIPA or NP-40 buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if phosphorylation status is important

  • Sonicate briefly to shear genomic DNA

• Gel separation:

  • Use 8-10% SDS-PAGE gels to properly resolve the 71-82 kDa HNRNPR protein

  • Load 20-50 μg of total protein per lane

• Transfer conditions:

  • Wet transfer at 100V for 1-2 hours or 30V overnight at 4°C

  • Use PVDF membrane for better protein retention

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary HNRNPR antibody at 1:500-1:2000 dilution overnight at 4°C

  • Use GAPDH or other appropriate loading controls (as shown in the Abcam validation data)

• Detection:

  • Employ HRP-conjugated secondary antibodies and enhanced chemiluminescence

  • Expected band size: 71-82 kDa (observed at 78 kDa in HEK-293T cells)

Optimization of antibody concentration for each specific lot and experimental system is recommended.

How can HNRNPR antibodies be used to study RNA-protein interactions?

HNRNPR antibodies can be effectively used to investigate RNA-protein interactions through several advanced methodologies:

• RNA Immunoprecipitation (RIP):

  • Cross-link RNA-protein complexes in vivo using UV or formaldehyde

  • Lyse cells and immunoprecipitate using HNRNPR antibody

  • Extract bound RNAs and analyze by qPCR or sequencing

  • This approach successfully identified 7SK RNA interaction with HNRNPR

• iCLIP (individual-nucleotide resolution Cross-Linking and Immunoprecipitation):

  • Cross-link RNA-protein complexes in living cells

  • Partially digest RNA, immunoprecipitate with HNRNPR antibody

  • Convert RNA to cDNA and sequence

  • This technique revealed HNRNPR binding to 3' UTR of mRNAs

• In vitro binding assays:

  • Combine recombinant His-tagged HNRNPR with candidate RNAs

  • Immunoprecipitate using anti-His antibody

  • Analyze co-precipitated RNAs by qPCR

  • This approach confirmed direct binding between HNRNPR and 7SK RNA

These methodologies have revealed that HNRNPR binds to specific structural elements like stem-loops in target RNAs, providing insights into its functional mechanisms.

What methodology should be used to investigate HNRNPR's role in mRNA splicing?

To investigate HNRNPR's function in mRNA splicing, researchers should employ a multi-faceted approach:

• Splicing reporter assays:

  • Construct minigene splicing reporters containing exons and introns of interest

  • Manipulate HNRNPR levels through knockdown or overexpression

  • Analyze splicing outcomes using RT-PCR and sequencing

• RNA-seq after HNRNPR modulation:

  • Perform HNRNPR knockdown or overexpression in relevant cell types

  • Conduct RNA-seq to identify global splicing alterations

  • Analyze for intron retention, exon skipping, and alternative splice site usage

• In situ splicing analysis:

  • Use HNRNPR antibodies for co-immunoprecipitation of spliceosome components

  • Identify associating splicing factors and RNA targets

  • Correlate findings with functional outcomes in splicing assays

• Domain-specific functional analysis:

  • Create domain deletion constructs of HNRNPR

  • Test their effects on splicing of target transcripts

  • Identify which structural domains mediate specific splicing functions

These approaches can reveal both direct and indirect effects of HNRNPR on pre-mRNA processing and specific splicing events in different cellular contexts.

How can researchers study the relationship between HNRNPR and 7SK noncoding RNA?

The interaction between HNRNPR and 7SK noncoding RNA represents an important area of research that can be investigated through these methodological approaches:

• Structure-function analysis of binding interfaces:

  • Generate deletion mutants of 7SK RNA, particularly in stem-loop regions (SL1 and SL3)

  • Perform in vitro binding assays with recombinant HNRNPR

  • Quantify binding efficiency through RNA immunoprecipitation followed by qPCR

  • Results have shown that deletions in SL1 and SL3 significantly reduce 7SK binding to HNRNPR

• In vivo validation of interactions:

  • Create knockdown constructs expressing shRNA targeting 7SK

  • Co-express 7SK deletion mutants resistant to knockdown

  • Perform RNA immunoprecipitation of HNRNPR

  • Analyze relative binding of 7SK mutants compared to endogenous 7SK

• Functional consequence assessment:

  • Investigate how HNRNPR-7SK interactions affect:

    • Transcriptional regulation

    • P-TEFb activity

    • RNA polymerase II pause release

  • Compare cellular phenotypes when disrupting HNRNPR-7SK binding versus disrupting other 7SK interactions

These approaches have revealed that HNRNPR binding to 7SK requires specific structural elements, particularly stem-loops SL1 and SL3, with both being necessary for optimal interaction .

What controls are essential when using HNRNPR antibodies in immunoprecipitation experiments?

For reliable immunoprecipitation (IP) experiments using HNRNPR antibodies, researchers should implement these essential controls:

• Negative IP controls:

  • Non-specific IgG antibody from the same species as the HNRNPR antibody

  • Lysate-only samples processed without antibody addition

  • HNRNPR knockout or knockdown cell lysates (when available)

• Input controls:

  • Analyze 5-10% of pre-IP lysate for baseline target abundance

  • Use for normalization of IP efficiency across samples

• Antibody validation controls:

  • When possible, use two different HNRNPR antibodies targeting distinct epitopes

  • For example, one antibody specific for the C-terminus (detecting both isoforms) and another specific for the N-terminus of the longer isoform

• Technical and biological replication:

  • Perform at least three technical replicates

  • Validate key findings with additional biological replicates

• Specificity controls for RNA-IP experiments:

  • Include control RNAs not expected to interact with HNRNPR (e.g., GAPDH mRNA)

  • Include antisense RNA controls for structured RNA interactions

  • Analyze background binding to beads or non-specific antibodies

How should researchers address inconsistent Western blot results with HNRNPR antibodies?

When encountering inconsistent Western blot results with HNRNPR antibodies, consider these methodological solutions:

• Protein extraction optimization:

  • HNRNPR is located in both nuclear and cytoplasmic compartments

  • Ensure complete lysis with appropriate buffers (RIPA or NP-40 with brief sonication)

  • For complete extraction, consider preparing separate nuclear and cytosolic fractions

• Post-translational modifications:

  • HNRNPR can exhibit different molecular weights due to post-translational modifications

  • Expected molecular weight is 71kDa, but often observed at 78-82kDa

  • Use denaturing conditions that preserve these modifications

• Antibody selection and dilution:

  • HNRNPR has multiple isoforms; select antibodies that recognize relevant epitopes

  • Optimize antibody dilution (typically 1:500-1:2000)

  • Consider using knockout-validated antibodies when available

• Transfer conditions:

  • Optimize transfer time and voltage for proteins >70kDa

  • Wet transfer is generally more effective than semi-dry for larger proteins

• Detection sensitivity:

  • Use enhanced chemiluminescence substrates appropriate for low-abundance proteins

  • Consider longer exposure times if signal is weak

When troubleshooting, methodically change one variable at a time and document all modifications to identify the source of inconsistency.

What are the key considerations for detecting endogenous versus recombinant HNRNPR?

Detection of endogenous versus recombinant HNRNPR presents distinct methodological challenges:

• Epitope accessibility differences:

  • Recombinant proteins may have different folding patterns affecting epitope exposure

  • Consider using antibodies recognizing different epitopes for validation

  • For recombinant proteins with tags, use tag-specific antibodies as alternative detection method

• Expression level considerations:

  • Recombinant HNRNPR is typically overexpressed compared to endogenous levels

  • Adjust antibody dilutions accordingly (more dilute for recombinant detection)

  • Use shorter exposure times for recombinant protein detection

• Molecular weight differences:

  • Endogenous HNRNPR appears at 71-82kDa

  • Recombinant proteins with tags will have altered molecular weights

  • Account for weight contributed by fusion tags (His, GST, etc.)

• Isoform specificity:

  • Consider which HNRNPR isoform is being expressed recombinantly

  • Choose antibodies that recognize either specific isoforms or all isoforms

  • N-terminal antibodies may not detect all isoforms

• Background concerns:

  • Higher background is common when detecting overexpressed proteins

  • Optimize blocking conditions and washing steps

  • Consider more stringent washing buffers for recombinant protein detection

These considerations help ensure accurate detection and differentiation between endogenous and recombinant HNRNPR in experimental systems.

How can researchers distinguish between different HNRNPR isoforms?

Distinguishing between HNRNPR isoforms requires specific methodological approaches:

• Isoform-specific antibody selection:

  • Use antibodies targeting the N-terminus to specifically detect the longer isoform

  • Use C-terminal antibodies to detect both isoforms simultaneously

  • As demonstrated in the literature, antibody "Ab2" specifically recognizes the N-terminus of the longer isoform, while "Ab1" identifies both long and short isoforms

• Gel electrophoresis optimization:

  • Use lower percentage gels (6-8%) for better separation of closely-sized isoforms

  • Consider using gradient gels for improved resolution

  • Extend running time to enhance separation of similar molecular weight bands

• RT-PCR for transcript identification:

  • Design primers flanking exon 2 (which is alternatively spliced)

  • Perform RT-PCR to detect presence of both transcript variants

  • Use quantitative RT-PCR to determine relative abundance of each isoform

• Mass spectrometry validation:

  • Immunoprecipitate HNRNPR using antibodies that detect both isoforms

  • Analyze by mass spectrometry to identify isoform-specific peptides

  • Quantify relative abundance of isoform-specific peptides

• Isoform-specific knockdown:

  • Design siRNAs targeting unique regions of each isoform

  • Confirm specific knockdown by Western blot using isoform-specific antibodies

  • Evaluate functional consequences of individual isoform depletion

These approaches enable precise identification and functional characterization of specific HNRNPR isoforms in different experimental contexts.

What are the emerging applications of HNRNPR antibodies in disease research?

HNRNPR antibodies are increasingly valuable tools in disease research, particularly in:

• Cancer research:

  • Investigating HNRNPR expression levels in different cancer types

  • Examining correlations between HNRNPR expression and patient outcomes

  • Studying the role of HNRNPR in regulating cancer-related gene expression and RNA processing

• Neurodegenerative disorders:

  • Exploring HNRNPR's role in RNA metabolism in neuronal cells

  • Investigating aberrant RNA processing in conditions like ALS or Alzheimer's

  • Examining HNRNPR interactions with disease-associated RNAs and proteins

• Autoimmune disease research:

  • Studying autoantibodies against HNRNPR in conditions like connective tissue diseases

  • Investigating concomitant autoimmune responses to HNRNPR and other nuclear antigens

  • The relationship between HNRNPR autoantibodies and disease pathogenesis has been documented

• Viral infection studies:

  • Examining how HNRNPR regulates viral RNA processing

  • Investigating HNRNPR's role in viral replication cycles

  • Developing potential therapeutic approaches targeting HNRNPR-viral RNA interactions

These emerging applications highlight the importance of HNRNPR antibodies as tools for understanding disease mechanisms and identifying potential therapeutic targets.

How should researchers interpret HNRNPR localization data in different cell types?

When interpreting HNRNPR localization data across different cell types, researchers should consider these methodological principles: