RPRD1B Antibody

What is RPRD1B and what role does it play in cellular processes?

RPRD1B (Regulation of Nuclear Pre-mRNA Domain Containing 1B) is a 37 kDa protein that functions as an RNA Polymerase II C-Terminal Domain (CTD) interacting protein . It contains a CTD-Interacting Domain (CID) that recognizes and binds to specific phosphorylation patterns on the CTD of RNA Polymerase II . RPRD1B plays critical roles in transcriptional regulation by promoting chromatin looping of genes such as CCND1 (cyclin D1), thereby recycling RNA Polymerase II from termination sites back to promoter regions . This recycling mechanism facilitates continuous rounds of transcription and can contribute to tumorigenesis when dysregulated . Additionally, RPRD1B regulates transcription of various genes through associations with STAT3 and the histone acetyltransferase p300 .

What structural domains are present in RPRD1B and how do they function?

RPRD1B contains several functional domains with specific roles:

• CTD-Interacting Domain (CID): Located at the N-terminus (amino acids 2-135), this domain forms a right-handed superhelical structure with 8 α-helices that create a concave channel for binding to the RNA Pol II CTD .

• Coiled-coil domain: Located at positions 171-304, this domain likely facilitates protein-protein interactions .

• RNA recognition regions: These enable RPRD1B to interact with RNA species during transcriptional processes .

The CID domain is particularly important as it contains conserved residues (R114, D65, R106) that mediate binding to the CTD of RNA Polymerase II with different specificities depending on the phosphorylation state of the CTD . Crystal structures have revealed that the RPRD1B CID can form domain-swapped dimers that may be stabilized by disulfide bonds involving C100 residues .

What types of RPRD1B antibodies are available for research purposes?

Several types of RPRD1B antibodies are available for research applications:

• Polyclonal rabbit antibodies: These recognize multiple epitopes on RPRD1B and are useful for general detection applications .

• Monoclonal mouse antibodies: These include antibodies raised against full-length recombinant RPRD1B (amino acids 1-326), offering high specificity for targeted epitopes .

• Antibodies targeting specific regions:

  • N-terminal region antibodies (AA 2-172)

  • C-terminal region antibodies (AA 254-282, AA 261-290)

  • Middle region antibodies (AA 143-192)

• Species-specific reactivity: Most commercially available antibodies have reactivity against human RPRD1B, while some also cross-react with mouse, rat, cow, dog, and other species .

What applications are RPRD1B antibodies validated for?

RPRD1B antibodies have been validated for multiple research applications:

When selecting an RPRD1B antibody, researchers should consider the specific application requirements and choose antibodies validated for their intended use with appropriate species reactivity .

How should I optimize Western blotting protocols when using RPRD1B antibodies?

For optimal Western blotting results with RPRD1B antibodies, follow these methodological considerations:

• Sample preparation:

  • Use freshly prepared lysates whenever possible

  • Include protease and phosphatase inhibitors to preserve protein integrity

  • Denature samples at 95-100°C for 5 minutes in reducing sample buffer

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution around 37 kDa (the molecular weight of RPRD1B)

  • Transfer to PVDF membranes at 100V for 1 hour or 30V overnight for efficient transfer

• Antibody incubation:

  • Block membranes with 5% non-fat dry milk or BSA in TBST

  • Dilute primary RPRD1B antibody to 1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash thoroughly (4-5 times for 5 minutes each) with TBST

• Detection:

  • Use HRP-conjugated secondary antibodies specific to the host species of your primary antibody

  • Visualize using ECL detection reagents

  • Expected band: 37 kDa for endogenous RPRD1B

Including positive controls (cell lines known to express RPRD1B) and negative controls (RPRD1B knockdown samples) will help validate specificity of detection .

What controls should I include when studying RPRD1B-CTD interactions?

When investigating RPRD1B interactions with RNA Polymerase II CTD, include these essential controls:

• Binding specificity controls:

  • R114A mutant RPRD1B: This mutation abolishes binding to RNA Polymerase II and can serve as a negative control

  • Q20A or R72A mutants: These mutations have minimal effect on binding and can serve as positive controls

• Phosphorylation state controls:

  • Unmodified CTD peptides

  • S2P-modified CTD peptides

  • S7P-modified CTD peptides

  • S5P-modified CTD peptides (which interfere with binding)

• Immunoprecipitation controls:

  • Input samples (5-10% of starting material)

  • IgG control immunoprecipitations

  • Antibodies against total RNA Polymerase II (e.g., N20)

  • Antibodies against specific phosphoisoforms of RNA Polymerase II CTD

These controls will help distinguish specific from non-specific interactions and validate the phosphorylation-dependent binding characteristics of RPRD1B to RNA Polymerase II CTD .

How does RPRD1B recognize different phosphorylation states of the RNA Polymerase II CTD?

RPRD1B exhibits differential binding preferences for various phosphorylation states of the RNA Polymerase II CTD through specific structural interactions:

The differential binding affinities (determined by isothermal titration calorimetry) are as follows:

These differential interactions likely enable RPRD1B to recognize specific phases of the transcription cycle based on the phosphorylation code of the RNA Pol II CTD .

What structural features enable RPRD1B to form functional complexes with RNA Polymerase II?

The structural elements that enable RPRD1B to form functional complexes with RNA Polymerase II include:

• CID domain architecture: The CID forms a right-handed superhelical structure with 8 α-helices arranged to create a concave channel that accommodates the CTD in a linear conformation . This channel positions key residues to interact with specific amino acids in the CTD heptapeptide repeats.

• Critical binding residues:

  • R114: Essential for binding to all CTD forms regardless of phosphorylation state

  • D65: Important for general CTD binding

  • R106: Critical for specific recognition of S2P modification

  • N69: Involved in water-mediated interactions with S7 hydroxyl groups

• Domain swapping capabilities: Crystal structures reveal that RPRD1B CID can form domain-swapped dimers stabilized by disulfide bonds involving C100 residues . While the biological significance of this configuration remains unclear, it may represent a regulatory mechanism for RPRD1B function.

• Coiled-coil domain: The C-terminal coiled-coil domain (residues 171-304) likely facilitates protein-protein interactions that may stabilize RPRD1B-CTD complexes or recruit additional factors to transcription sites .

These structural features collectively enable RPRD1B to recognize the RNA Pol II CTD in a phosphorylation-dependent manner and to participate in transcriptional regulation by facilitating chromatin looping and polymerase recycling .

Why might I observe inconsistent results when detecting RPRD1B in different cell types?

Inconsistent detection of RPRD1B across different cell types may result from several factors:

• Expression level variations: RPRD1B expression can vary significantly between cell types and tissues, with higher expression often observed in proliferating cells due to its role in regulating cyclin D1 transcription .

• Post-translational modifications: RPRD1B may undergo various post-translational modifications that affect antibody recognition or protein mobility on gels. These modifications might vary depending on cell type or cellular conditions.

• Protein-protein interactions: RPRD1B forms complexes with RNA Polymerase II, STAT3, and p300 , which may mask epitopes or alter antibody accessibility in different cellular contexts.

• Antibody specificity issues: Some antibodies may cross-react with RPRD1A or RPRD2, which share structural similarities, particularly in the CID region . The sequence homology between these proteins can lead to detection of multiple bands.

• Isoform differences: Alternative splicing could generate RPRD1B isoforms that differ between cell types, potentially resulting in unexpected banding patterns if an antibody's epitope spans a splice junction.

To address these issues, validate results using multiple antibodies targeting different epitopes of RPRD1B and include appropriate positive controls from cell types known to express RPRD1B at detectable levels.

How can I verify the specificity of my RPRD1B antibody?

To verify RPRD1B antibody specificity, implement these methodological approaches:

• Genetic validation:

  • siRNA or shRNA knockdown of RPRD1B should reduce or eliminate the specific band at 37 kDa

  • CRISPR/Cas9 knockout cells provide definitive negative controls

  • Overexpression of tagged RPRD1B can serve as a positive control

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • The specific RPRD1B signal should be significantly reduced or eliminated

• Cross-reactivity testing:

  • Test the antibody against purified recombinant RPRD1A, RPRD1B, and RPRD2 proteins

  • A specific antibody should recognize only RPRD1B and not related family members

• Mutation-based validation:

  • Test antibody recognition of wild-type RPRD1B versus mutants (such as R114A)

  • If the epitope includes the mutated region, altered binding may occur

• Multiple application validation:

  • Confirm consistent results across different applications (WB, IP, IHC)

  • Correlate results between different detection methods

For research requiring absolute specificity, combining these approaches provides the most robust validation of RPRD1B antibody performance and ensures reliable experimental results.

How can RPRD1B antibodies be used to study transcriptional regulation mechanisms?

RPRD1B antibodies enable comprehensive investigation of transcriptional regulation mechanisms through these methodological approaches:

• Chromatin immunoprecipitation (ChIP):

  • Use RPRD1B antibodies to identify genomic binding sites

  • Combine with sequencing (ChIP-seq) to establish genome-wide binding profiles

  • Correlation with RNA Pol II phosphoisoform ChIP data can reveal transcriptional state-specific associations

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate RPRD1B to identify interacting partners such as RNA Pol II, STAT3, and p300

  • Use different CTD phosphoisoform-specific antibodies to determine which transcription states associate with RPRD1B

  • Map interaction domains through mutational analysis (e.g., R114A mutant abolishes RNA Pol II binding)

• Chromatin conformation capture (3C/4C/Hi-C):

  • Combined with RPRD1B ChIP to study its role in chromatin looping

  • Particularly useful for examining RPRD1B's function in recycling Pol II from termination sites to promoters at genes like CCND1

• Transcriptional pulse-chase experiments:

  • Track RNA Pol II progression through genes in the presence or absence of RPRD1B

  • Determine how RPRD1B affects transcription elongation rates and RNA processing

Each of these approaches provides unique insights into RPRD1B's function in transcriptional regulation and can be enhanced by using highly specific antibodies against RPRD1B and its interacting partners.

What are emerging approaches for studying RPRD1B function in disease models?

Emerging research approaches using RPRD1B antibodies in disease models include:

• Cancer research applications:

  • Immunohistochemistry (IHC) to correlate RPRD1B expression with tumor progression

  • Tissue microarray analysis to examine RPRD1B levels across cancer subtypes

  • Investigation of RPRD1B's role in regulating cyclin D1 transcription and tumorigenesis

• Proximity-based labeling techniques:

  • BioID or APEX2 fusions with RPRD1B to identify protein interaction networks in normal versus disease states

  • Mapping spatial organization of RPRD1B-containing complexes at promoters versus termination sites

• Live-cell imaging approaches:

  • Combine antibody-based detection with super-resolution microscopy to visualize RPRD1B dynamics during transcription

  • FRAP (Fluorescence Recovery After Photobleaching) to measure RPRD1B turnover rates at active genes

• Therapeutic target validation:

  • Using antibodies to evaluate RPRD1B expression changes in response to cancer therapies

  • Screening for compounds that disrupt pathological RPRD1B interactions, particularly its association with the CCND1 promoter

• Mouse models:

  • Antibody-based validation of RPRD1B knockout or transgenic mouse models

  • Immunohistochemical analysis of tissue-specific RPRD1B expression patterns during development and disease progression

These approaches leverage the specificity of RPRD1B antibodies to understand its role in disease mechanisms and potentially identify new therapeutic strategies targeting RPRD1B-mediated transcriptional dysregulation.