PRPF19 Antibody

What is PRPF19 and what are its primary biological functions?

PRPF19 (also known as PRP19, PSO4, SNEV, or UBOX4) is a ubiquitin-protein ligase that functions as a core component of several complexes primarily involved in pre-mRNA splicing and DNA repair. PRPF19 serves multiple critical cellular functions:

• Pre-mRNA splicing: PRPF19 is a required component of the spliceosome, participating in its assembly and remodeling. It mediates 'Lys-63'-linked polyubiquitination of the U4 spliceosomal protein PRPF3, which allows recognition by the U5 component PRPF8 and stabilizes the U4/U5/U6 tri-snRNP spliceosomal complex .

• DNA damage response: As part of the PRP19-CDC5L complex, PRPF19 is recruited to DNA damage sites by the RPA complex, where it directly ubiquitinates RPA1 and RPA2. This 'Lys-63'-linked polyubiquitination enables recruitment of the ATR-ATRIP complex and activation of ATR, a master regulator of DNA damage response .

• DNA repair: PRPF19 may play roles in DNA double-strand break repair by recruiting repair factors to altered DNA and in DNA interstrand cross-links repair as part of the PSO4 complex .

• Ubiquitination and protein degradation: PRPF19 can mediate 'Lys-48'-linked polyubiquitination of substrates, potentially playing a role in proteasomal degradation .

Recent research has also implicated PRPF19 in cancer progression, cellular senescence, and antiviral defense mechanisms .

What types of PRPF19 antibodies are available for research?

Several types of PRPF19 antibodies are available for research applications:

Most commercially available PRPF19 antibodies are generated using recombinant fusion proteins or specific peptide sequences. For example, the polyclonal antibody CAB12590 is generated against a recombinant fusion protein containing amino acids 127-416 of human PRPF19 (NP_055317.1) .

How does PRPF19 expression vary across different tissues and cell types?

PRPF19 expression has been documented in various tissues and cell types, with notable variations in expression patterns:

• Cancer tissues: Elevated PRPF19 expression has been observed in bladder urothelial carcinoma (BLCA) compared to normal bladder tissues, suggesting its potential as a prognostic biomarker .

• Immune cells: Single-cell RNA sequencing data from the TISCH database reveals that PRPF19 is expressed in actively proliferating CD4 Tconv, CD8T, and NK cells within bladder cancer microenvironments .

• Cell lines: PRPF19 protein has been detected in various cell lines including HEK-293, HeLa, PC-3, and Jurkat cells, making these suitable positive controls for antibody validation .

For immunohistochemistry studies, human stomach tissue has shown positive staining with PRPF19 antibodies .

What are the optimal dilution ratios for different applications of PRPF19 antibodies?

The optimal dilution ratios for PRPF19 antibodies vary by application and specific antibody. Based on the search results, the following dilutions are recommended:

For the Proteintech PRPF19 antibody (15414-1-AP), it is specifically recommended to use a dilution of 1:500-1:2000 for Western blot and 1:50-1:500 for immunohistochemistry .

It is crucial to optimize the dilution for each specific experimental system to obtain optimal results, as sensitivity may vary with different sample types and detection methods .

What protocols are recommended for immunohistochemistry with PRPF19 antibodies?

For immunohistochemistry (IHC) with PRPF19 antibodies, the following protocol based on search results is recommended:

• Tissue preparation: Dewax the microarray or tissue sections using standard procedures.

• Antigen retrieval:

  • Primary recommendation: Use TE buffer pH 9.0 for antigen retrieval .

  • Alternative method: Citrate buffer pH 6.0 can also be used .

  • Apply endogenous peroxidase blocker dropwise to the tissue and incubate at room temperature for 10 minutes for antigen repair .

• Blocking: Complete appropriate blocking step based on your detection system.

• Primary antibody incubation:

  • Apply PRPF19 antibody at the recommended dilution (1:50-1:200) .

  • Incubate overnight at 4°C .

• Secondary antibody incubation:

  • Wash with appropriate buffer.

  • Incubate with compatible secondary antibody for 30 minutes .

• Color development and counterstaining:

  • Develop color using an appropriate substrate.

  • Counterstain, dehydrate, and mount.

• Scoring (if quantification is needed):

  • Staining intensity can be categorized as low, medium, and high (assigned scores of 1, 2, 3).

  • Staining range can be categorized as 0-25%, 26%-50%, 51%-75%, and 76%-100% (assigned scores of 1, 2, 3, and 4).

  • Multiply the two scores to obtain a total score, with ≤6 being low expression and >6 being high expression .

For validation, human stomach tissue has been demonstrated as positive control tissue for PRPF19 IHC staining .

What is the recommended protocol for Western blot analysis using PRPF19 antibodies?

Based on the search results, the following protocol is recommended for Western blot analysis using PRPF19 antibodies:

• Sample preparation: Prepare cell or tissue lysates using standard protocols. Several positive controls have been validated including:

  • Cell lysates: HEK-293, HeLa, PC-3, and Jurkat cells

  • Tissue lysates: Mouse kidney, mouse liver, rat kidney, and human testis

• Protein separation:

  • Perform SDS-PAGE to separate proteins

  • PRPF19 has a calculated molecular weight of approximately 55 kDa and is typically observed at 55 kDa on Western blots

• Transfer and blocking:

  • Transfer proteins to a membrane (PVDF or nitrocellulose)

  • Block with appropriate blocking buffer

• Antibody incubation:

  • Primary antibody: Dilute PRPF19 antibody at 1:500-1:2000 in appropriate buffer

  • Incubate according to the antibody manufacturer's recommendations

  • Secondary antibody: Use appropriate HRP-conjugated or fluorescently-labeled secondary antibody

• Detection:

  • Develop using chemiluminescence or fluorescence detection systems

  • PRPF19 should be detected at approximately 55 kDa

For monitoring PRPF19 expression in experimental studies (such as overexpression or knockdown), appropriate controls should be included to validate the specificity of detected bands .

How can I validate the specificity of PRPF19 antibodies in my experimental system?

To validate the specificity of PRPF19 antibodies in your experimental system, consider implementing the following approaches:

• Positive and negative controls:

  • Use cell lines with known PRPF19 expression as positive controls (e.g., HEK-293, HeLa, PC-3, Jurkat cells)

  • Include tissues with validated PRPF19 expression (e.g., mouse kidney, human testis)

  • For negative controls, consider using PRPF19 knockdown cells or tissues

• Knockdown/knockout validation:

  • Perform siRNA-mediated knockdown or CRISPR-Cas9 knockout of PRPF19

  • Compare antibody signals between wild-type and knockout/knockdown samples

  • A specific antibody should show significantly reduced or absent signal in knockdown/knockout samples

• Overexpression validation:

  • Express tagged PRPF19 (e.g., FLAG-PRPF19) in cells

  • Perform parallel detection with anti-PRPF19 and anti-tag antibodies

  • Co-localization or similar pattern of signal increase confirms specificity

• Molecular weight confirmation:

  • PRPF19 has a calculated molecular weight of 55 kDa

  • Verify that your antibody detects a band at the expected size in Western blot

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide/protein

  • A specific antibody's signal should be blocked or significantly reduced

For published studies, researchers have validated PRPF19 antibodies by analyzing both protein and mRNA levels following PRPF19 overexpression or knockdown, observing corresponding changes in antibody signal intensity .

What are common troubleshooting issues with PRPF19 antibodies and how can they be resolved?

Based on the available search results and general antibody troubleshooting principles, here are common issues with PRPF19 antibodies and potential solutions:

• Weak or no signal in Western blot:

  • Increase antibody concentration (try 1:500 instead of 1:2000)

  • Increase protein loading amount

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced detection systems

  • Verify sample preparation (ensure protein is not degraded)

  • Check if denaturation conditions are appropriate for epitope exposure

• High background in immunohistochemistry/immunocytochemistry:

  • Optimize antibody dilution (try more diluted solutions, e.g., 1:200 instead of 1:50)

  • Improve blocking conditions (extend blocking time or try different blocking agents)

  • Reduce primary antibody incubation time

  • Increase washing steps duration and number

  • For IHC, try different antigen retrieval methods (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Multiple bands in Western blot:

  • Verify if bands represent isoforms, degradation products, or post-translational modifications

  • Increase stringency of washing steps

  • Use fresh samples to minimize degradation

  • Include protease inhibitors during sample preparation

  • Validate with PRPF19 knockdown to determine which band represents specific signal

• Variable results across experiments:

  • Standardize protocols, including sample preparation, antibody dilutions, and incubation times

  • Use consistent positive controls across experiments

  • Store antibodies according to manufacturer recommendations (typically at -20°C with 50% glycerol)

  • Avoid repeated freeze-thaw cycles

  • Aliquot antibodies for single use

• Poor immunoprecipitation efficiency:

  • Optimize antibody amount (try 0.5μg-4μg antibody for 200μg-400μg extracts)

  • Adjust lysis conditions to preserve protein interactions

  • Extend incubation time with the antibody

  • Consider using protein A/G beads instead of agarose if efficiency is low

For storing PRPF19 antibodies, manufacturers recommend -20°C for long-term storage (stable for one year). For frequent use, 4°C storage for up to one month is acceptable, but repeated freeze-thaw cycles should be avoided .

How can I design experiments to study PRPF19's role in the ubiquitination pathway?

To investigate PRPF19's role in the ubiquitination pathway, consider the following experimental design approaches based on the search results:

• Protein interaction studies:

  • Co-immunoprecipitation: Use PRPF19 antibodies to pull down PRPF19 and its associated proteins. Western blot analysis can then identify ubiquitination-related proteins that interact with PRPF19 .

  • Proximity ligation assay: Detect in situ protein-protein interactions between PRPF19 and suspected ubiquitination targets or adaptors.

• Ubiquitination assays:

  • In vivo ubiquitination: Overexpress PRPF19 along with tagged ubiquitin (e.g., HA-Ub) and potential substrate proteins. Immunoprecipitate the substrate and immunoblot for ubiquitin to detect PRPF19-mediated ubiquitination .

  • In vitro ubiquitination: Use purified components (E1, E2, PRPF19 as E3, ubiquitin, and substrate) to reconstitute the ubiquitination reaction in a test tube.

• Functional studies:

  • Mutational analysis: Generate PRPF19 mutants lacking E3 ligase activity to compare with wild-type PRPF19.

  • Structure-function analysis: Create domain deletions to identify regions critical for ubiquitination activity.

• Substrate identification:

  • Proteomics approach: Combine PRPF19 overexpression/knockdown with ubiquitin proteomics to identify differentially ubiquitinated proteins.

  • Candidate approach: Test potential substrates based on known PRPF19 functions or interactions.

• Pathway analysis:

  • PRPF19-mediated degradation: As demonstrated in the study of ATXN3-polyQ protein, measure substrate protein levels upon PRPF19 overexpression or knockdown to determine if PRPF19 promotes degradation .

  • Ubiquitin chain analysis: Determine the type of ubiquitin chains (K48, K63, etc.) formed by PRPF19 using chain-specific antibodies.

An example from the search results is the study of PRPF19's role in degrading expanded ATXN3-polyQ protein:

• Researchers first established physical interaction between PRPF19 and polyQ protein using co-immunoprecipitation

• They then demonstrated that PRPF19 overexpression reduced levels of ATXN3-Q71 and suppressed caspase-3 activation

• Conversely, knockdown of PRPF19 led to increased ATXN3-Q71 levels and enhanced caspase-3 cleavage

• These effects were specific to expanded polyQ proteins (ATXN3-Q71) and not observed with unexpanded proteins (ATXN3-Q28)

How can PRPF19 antibodies be used to investigate its role in cancer progression?

Based on the search results, PRPF19 antibodies can be effectively employed to investigate its role in cancer progression through several sophisticated approaches:

• Expression analysis in cancer tissues:

  • Tissue microarrays: Use immunohistochemistry with PRPF19 antibodies to analyze expression patterns across multiple cancer samples. In bladder cancer studies, researchers used tissue microarrays containing 56 bladder cancer tissues and 32 normal bladder tissues to assess PRPF19 expression levels .

  • Scoring systems: Implement quantitative scoring methods where staining intensity (low, medium, high; scores 1-3) and staining range (0-25%, 26-50%, 51-75%, 76-100%; scores 1-4) are multiplied to obtain a total score. Scores ≤6 can be classified as low expression while >6 as high expression .

• Correlation with clinicopathological features:

  • Use PRPF19 antibodies to stratify cancer tissues based on expression levels, then correlate with:

    • Tumor grade (low vs. high grade)

    • Clinical stage

    • Patient survival outcomes

    • Metastatic status

  • Studies have shown PRPF19 expression differs between low-grade and high-grade bladder cancer samples .

• Functional studies in cancer cells:

  • Knockdown/overexpression: Modulate PRPF19 expression in cancer cells and measure effects on:

    • Proliferation

    • Migration/invasion

    • Apoptosis resistance

    • Drug sensitivity

  • Validate knockdown/overexpression efficiency using PRPF19 antibodies in Western blot

• Mechanistic investigations:

  • Immune microenvironment analysis: Single-cell RNA sequencing combined with PRPF19 antibody-based flow cytometry can reveal cell-specific expression patterns. Research has shown elevated PRPF19 expression in actively proliferating CD4 Tconv, CD8T, and NK cells within bladder cancer microenvironments .

  • Pathway analysis: Use PRPF19 antibodies in combination with antibodies against pathway components to investigate mechanisms. Gene set enrichment analysis has linked PRPF19 to several cancer-relevant pathways including:

    • DNA methylation

    • Senescence-associated secretory phenotype

    • Cell cycle regulation

    • E2F pathway

• Prognostic biomarker validation:

  • Perform survival analysis comparing patient outcomes based on PRPF19 expression levels detected by immunohistochemistry

  • Transcriptomic data and bladder cancer tissue microarrays have identified high expression of PRPF19 in bladder urothelial carcinoma (BLCA), suggesting its potential as a prognostic biomarker

What methods can be used to study PRPF19's role in DNA damage response?

To investigate PRPF19's role in DNA damage response, researchers can employ several sophisticated methods:

• Localization studies after DNA damage induction:

  • Immunofluorescence microscopy: Use PRPF19 antibodies to track protein localization before and after DNA damage induced by:

    • UV irradiation

    • Ionizing radiation

    • Radiomimetic drugs (e.g., bleomycin, etoposide)

    • Crosslinking agents (e.g., mitomycin C)

  • Co-localization analysis: Double staining with PRPF19 antibodies and antibodies against known DNA damage markers (γH2AX, 53BP1, RAD51) to determine recruitment to damage sites

• Protein-protein interaction studies in the context of DNA damage:

  • Co-immunoprecipitation: Use PRPF19 antibodies to pull down PRPF19 complexes from cells before and after DNA damage, followed by Western blotting or mass spectrometry to identify damage-specific interactions

  • Proximity ligation assay: Detect in situ interactions between PRPF19 and DNA damage response proteins

  • Based on search results, PRPF19 is known to interact with the RPA complex at DNA damage sites and to ubiquitinate RPA1 and RPA2

• Functional studies:

  • PRPF19 depletion/overexpression: Modulate PRPF19 levels and assess effects on:

    • DNA damage checkpoint activation (ATR, CHK1 phosphorylation)

    • DNA repair kinetics (repair assays, comet assay)

    • Cell survival after damage (clonogenic assays)

    • Cell cycle checkpoints (flow cytometry)

  • Research has shown that 'Lys-63'-linked polyubiquitination of the RPA complex by PRPF19 allows recruitment of the ATR-ATRIP complex and activation of ATR

• Ubiquitination studies:

  • In vivo ubiquitination assays: Immunoprecipitate potential substrates (e.g., RPA1, RPA2) after DNA damage in cells with normal or altered PRPF19 levels, then immunoblot for ubiquitin

  • Chain-specific ubiquitin antibodies: Determine the type of ubiquitin chains formed (Lys-63 vs. Lys-48)

  • Studies indicate PRPF19 mediates 'Lys-63'-linked polyubiquitination in the DNA damage response context

• Repair pathway-specific assays:

  • DSB repair choice: Assess homologous recombination vs. non-homologous end joining using reporter assays in PRPF19-depleted cells

  • Interstrand crosslink repair: Measure sensitivity to crosslinking agents and repair kinetics

  • Research suggests PRPF19 may play a role in DNA double-strand break repair by recruiting the repair factor SETMAR to altered DNA and may be involved in DNA interstrand cross-links repair as part of the PSO4 complex

• Chromatin immunoprecipitation (ChIP):

  • Use PRPF19 antibodies to perform ChIP after DNA damage to identify genomic regions where PRPF19 is recruited

  • Combine with sequencing (ChIP-seq) for genome-wide analysis

These approaches can provide comprehensive insights into PRPF19's functions in the DNA damage response, building on the established knowledge that PRPF19 plays roles in both pre-mRNA splicing and DNA repair mechanisms.

How can researchers investigate PRPF19's role in antiviral defense mechanisms?

Based on the search results, researchers can employ several methodological approaches to investigate PRPF19's role in antiviral defense mechanisms:

• Viral infection models:

  • Cell culture systems: Establish appropriate cell lines (e.g., LLC-PK1 or Vero cells for PEDV studies) with modulated PRPF19 expression

  • Viral challenge assays: Infect cells with viruses of interest (such as porcine epidemic diarrhea virus/PEDV) and assess viral replication parameters

• PRPF19 expression manipulation:

  • Overexpression studies: Transfect cells with FLAG-PRPF19 expression constructs at varying concentrations to establish dose-dependent effects on viral replication

  • Silencing/knockdown approaches: Use siRNA or shRNA targeting PRPF19 to reduce its expression and observe effects on viral susceptibility

  • CRISPR-Cas9 knockout: Generate PRPF19-null cell lines for more complete functional analysis

• Viral replication assessment:

  • Quantitative RT-PCR: Measure viral mRNA levels (e.g., PEDV N mRNA) in cells with normal vs. altered PRPF19 expression

  • Western blot analysis: Detect viral protein levels (e.g., PEDV N protein) using specific antibodies

  • Viral titer determination: Perform TCID50 analysis to quantify infectious viral particles produced

  • Plaque assays: Visualize and quantify viral spread and replication capacity

• Mechanism investigation:

  • Co-immunoprecipitation: Use PRPF19 antibodies to identify interactions with viral components or host antiviral factors

  • Ubiquitination assays: Investigate whether PRPF19 ubiquitinates viral proteins for degradation

  • Autophagy-lysosome pathway analysis: Assess PRPF19's role in targeting viral components for degradation through this pathway

  • Confocal microscopy: Track co-localization of PRPF19 with viral components and cellular organelles

• Pathway analysis:

  • Transcriptomics: Compare gene expression profiles in infected cells with normal vs. altered PRPF19 levels

  • Signaling cascade analysis: Investigate the impact of PRPF19 on antiviral signaling pathways (e.g., interferon response)

  • Interaction with other host factors: Study PRPF19's cooperation with factors like MARCH8 and NDP52 in antiviral defense

Research has demonstrated that PRPF19 can suppress PEDV replication through a specific mechanism:

• PRPF19 degrades PEDV N protein through the autophagy-lysosome pathway

• This degradation is mediated by the E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52

• Overexpression of PRPF19 decreases both PEDV N protein and mRNA levels in a dose-dependent manner

• Silencing of PRPF19 enhances PEDV replication

These findings suggest PRPF19 functions as a novel antiviral protein, and similar methodological approaches could be applied to investigate its role against other viruses.

How should researchers interpret changes in PRPF19 expression in disease states?

Based on the search results, researchers should consider multiple layers of analysis when interpreting changes in PRPF19 expression in disease states:

• Expression level analysis:

  • Quantitative assessment: Use standardized scoring methods for IHC (multiplying intensity and distribution scores) to objectively compare PRPF19 expression between normal and diseased tissues

  • Statistical validation: Apply appropriate statistical tests (e.g., Wilcox test) to determine if differences in expression levels are significant

  • Multiple sample types: Compare expression across different sample types (e.g., primary tumors vs. metastases) to understand disease progression patterns

• Correlation with clinical parameters:

  • Survival analysis: Use Kaplan-Meier curves and log-rank tests to correlate PRPF19 expression with patient outcomes

  • Disease-specific parameters: For cancer studies, correlate PRPF19 expression with tumor grade, stage, and other relevant clinical features

  • Multivariate analysis: Determine if PRPF19 is an independent prognostic factor by controlling for other variables

• Functional interpretation:

  • Pathway enrichment analysis: Use gene set enrichment analysis (GSEA) to identify biological pathways associated with PRPF19 expression changes

  • Cell-type specific patterns: Analyze single-cell RNA sequencing data to understand cell-specific expression patterns of PRPF19 in disease microenvironments

  • Integration with other biomarkers: Consider how PRPF19 expression relates to other established disease markers

• Mechanistic insights:

  • Correlation with cellular processes: In bladder cancer, PRPF19 has been linked to specific processes including:

    • Immune cell infiltration and function

    • DNA methylation patterns

    • Cellular senescence and the senescence-associated secretory phenotype

    • Cell cycle regulation and the E2F pathway

  • Experimental validation: Use in vitro and in vivo models to verify mechanisms suggested by expression changes

• Biomarker potential assessment:

  • Predictive accuracy: Use time-dependent receiver operating characteristic (timeROC) analysis to assess the predictive value of PRPF19 expression

  • Sensitivity and specificity: Determine optimal cutoff values for classifying PRPF19 expression levels in disease diagnosis or prognosis

  • Comparison with standard markers: Evaluate if PRPF19 offers additional predictive value beyond established biomarkers

For example, in bladder urothelial carcinoma (BLCA):

What methodological approaches are recommended for analyzing PRPF19's interactions in regulatory networks?

Based on the search results, several methodological approaches are recommended for analyzing PRPF19's interactions in regulatory networks:

• Bioinformatic network analysis:

  • Construction of competing endogenous RNA (ceRNA) networks:

    • Use databases like MiRWALK to identify microRNAs that target PRPF19

    • Employ ENCORI to predict interactions between identified miRNAs and long non-coding RNAs (lncRNAs)

    • Analyze subcellular localization of network components using Genecards

    • Build networks based on ceRNA hypothesis (negative association between mRNAs/lncRNAs and miRNAs)

  • Validation through expression correlation:

    • Confirm predicted interactions by analyzing correlated expression in patient cohorts

    • Perform survival analysis to identify functionally significant interactions

• Machine learning approaches for network inference:

  • LASSO (Least Absolute Shrinkage and Selection Operator) method:

    • Select crucial immune cells or other interacting components from large datasets

    • Use LASSO to identify the most statistically relevant interactions

  • Predictive modeling:

    • Develop and validate predictive models based on PRPF19-associated networks

    • Assess model accuracy using methods such as time-dependent receiver operating characteristic (timeROC) analysis

• Experimental validation of network interactions:

  • RNA interference studies:

    • Knockdown PRPF19 or predicted interacting partners

    • Measure effects on expression of other network components

  • Overexpression studies:

    • Express PRPF19 and measure effects on predicted network components

    • Observe alterations in downstream pathways

  • Reporter assays:

    • Construct luciferase reporters containing predicted binding sites

    • Validate direct interactions between PRPF19-related miRNAs and target genes/lncRNAs

• Integration of multi-omics data:

  • Transcriptomics and proteomics correlation:

    • Analyze how PRPF19 mRNA and protein levels correlate with other network components

  • DNA methylation analysis:

    • Investigate the impact of DNA methylation on PRPF19 expression and network function using tools like the SMART website

  • Single-cell analysis:

    • Utilize single-cell data from databases like the Tumor Immunization Single Cell Center (TISCH) to understand cell-specific network operations

• Functional pathway analysis:

  • Gene set enrichment analysis (GSEA):

    • Identify pathways significantly associated with PRPF19 expression

    • Research has linked PRPF19 to several pathways including:

      • Immune response pathways (Sarscov2 innate immunity evasion, MHC pathway)

      • DNA methylation

      • Senescence-associated secretory phenotype

      • Cell cycle regulation

      • E2F pathway