RNF20 Antibody

What is RNF20 and what cellular functions does it regulate?

RNF20, also known as E3 ubiquitin-protein ligase BRE1A, is a 975 amino acid protein containing a RING-type zinc finger domain. It functions as a component of the RNF20/40 E3 ubiquitin-protein ligase complex that mediates monoubiquitination of histone H2B at lysine 120 (H2BK120ub1). This epigenetic modification is critical for transcriptional activation and serves as a prerequisite for histone H3 methylation at lysine 4 and lysine 79 positions. Through these mechanisms, RNF20 plays central roles in histone code regulation, gene expression control, transcriptional activation of Hox genes, and functions as a transcriptional coactivator when recruited to promoters such as MDM2 . RNF20 also mediates polyubiquitination of isoform 2 of PA2G4 in cancer cells, targeting it for proteasomal degradation . Recent research has demonstrated RNF20's involvement in RNA polymerase II promoter-proximal pausing, VEGFA signaling, and angiogenesis regulation .

What applications can RNF20 antibodies be used for in research settings?

RNF20 antibodies are validated for multiple research applications:

These applications allow researchers to detect endogenous RNF20 protein expression, localization, and interactions in various experimental systems . It is recommended to optimize antibody dilutions for each specific experimental system to obtain optimal results.

What species reactivity can be expected from commercially available RNF20 antibodies?

Available RNF20 antibodies demonstrate validated reactivity with multiple species, though this varies by manufacturer and antibody clone. Most commercially available antibodies show reactivity with human samples, with many also cross-reacting with mouse and rat proteins . Some antibodies have demonstrated reactivity with monkey samples . According to published literature and manufacturer validation data, certain antibodies have also been cited for use with pig samples . When selecting an antibody for a particular species, researchers should consult the specific product documentation for validated reactivity claims and consider validation testing in their model system before proceeding with large-scale experiments.

What are the critical considerations for optimizing RNF20 antibody performance in chromatin immunoprecipitation (ChIP) experiments?

While not explicitly listed in the standard applications for many commercial RNF20 antibodies, ChIP experiments are important for studying RNF20's role in histone modification and transcriptional regulation. For optimal ChIP performance, researchers should consider:

• Crosslinking optimization: Since RNF20 interacts with both histone H2B and transcriptional machinery, a dual crosslinking approach using both formaldehyde (protein-DNA) and protein-protein crosslinkers (DSG or EGS) may improve complex stability.

• Antibody selection: Choose antibodies validated for IP applications with demonstrated nuclear antigen recognition capability .

• Chromatin fragmentation: Optimize sonication conditions to generate 200-500bp fragments for highest resolution of RNF20 binding sites.

• Controls: Include IgG negative controls and positive controls targeting known RNF20-regulated loci such as HOX genes or MDM2 promoter regions .

• Sequential ChIP considerations: For studies examining the co-localization of RNF20 with H2BK120ub1 or other histone modifications, sequential ChIP protocols may require special optimization of elution conditions that don't denature the second target antigen.

How can researchers differentiate between active and inactive forms of RNF20 when using antibodies?

Distinguishing between active and inactive RNF20 poses a significant challenge in current research. RNF20's E3 ligase activity is regulated by post-translational modifications and protein-protein interactions rather than by dramatic conformational changes detectable by standard antibodies. To address this experimental challenge:

• Complement antibody detection with functional assays measuring H2B ubiquitination activity.

• Use phospho-specific antibodies if available, as phosphorylation may regulate RNF20 activity.

• Employ proximity ligation assays (PLA) to detect RNF20 interaction with known cofactors (RNF40, WAC, or UBE2A/B) as a proxy for activation state .

• Consider using ubiquitin-activated interaction trap (UBAIT) approaches to capture RNF20 in its catalytically active state.

• Combine RNF20 immunoprecipitation with subsequent activity assays using recombinant H2B as substrate to measure associated E3 ligase activity.

This multi-faceted approach can provide more comprehensive insights into RNF20 activation states than antibody detection alone.

What protocol modifications are necessary when using RNF20 antibodies for immunohistochemistry in different tissue types?

Optimal immunohistochemical detection of RNF20 requires tailored approaches for different tissue types:

For epithelial tissues:

• Standard antigen retrieval using TE buffer pH 9.0 has proven effective

• Dilution ranges of 1:50-1:200 typically yield optimal signal-to-noise ratios

For neural tissues:

• Extended antigen retrieval (15-20 minutes) may be necessary due to tissue density

• Lower antibody dilutions (1:50-1:100) often produce better results

For vascular tissues (particularly important given RNF20's role in angiogenesis):

• Citrate buffer (pH 6.0) can be used as an alternative antigen retrieval method

• Special attention to blocking endogenous peroxidase activity is essential

For all tissues, validation using appropriate positive controls (such as kidney tissue, which shows reliable RNF20 expression) and negative controls (including RNF20 knockdown samples if available) is strongly recommended . Additionally, researchers studying vascular development should consider dual immunofluorescence with endothelial markers to precisely localize RNF20 expression in the context of VEGFA signaling .

How should researchers troubleshoot weak or absent RNF20 signal in Western blot applications?

When encountering difficulties with RNF20 detection in Western blots, systematic troubleshooting should include:

• Sample preparation optimization:

  • Ensure complete nuclear protein extraction using specialized buffers containing 1% SDS or 8M urea

  • Include protease inhibitors to prevent degradation

  • Avoid multiple freeze-thaw cycles of lysates

• Protein transfer improvements:

  • For RNF20's high molecular weight (114 kDa), extend transfer time or reduce voltage

  • Consider semi-dry transfer systems for large proteins

  • Use PVDF membranes rather than nitrocellulose for better protein retention

• Antibody optimization:

  • Test multiple antibody concentrations between 1:1000-1:8000

  • Extend primary antibody incubation to overnight at 4°C

  • Consider testing multiple antibodies targeting different epitopes

• Detection enhancement:

  • Use enhanced chemiluminescence (ECL) substrates specifically designed for low-abundance proteins

  • Extend exposure times incrementally to capture weak signals

  • Consider fluorescent secondary antibodies for more quantitative detection

• Biological considerations:

  • Verify RNF20 expression in selected cell lines via public datasets

  • Utilize positive controls such as HeLa, MCF-7, or Jurkat cells with confirmed RNF20 expression

  • Consider using RNF20 overexpression samples as additional positive controls

What are the optimal storage and handling conditions to maintain RNF20 antibody performance over time?

To preserve RNF20 antibody functionality and prevent performance degradation:

• Storage temperature:

  • Store antibodies at -20°C as recommended by manufacturers

  • Maintain consistent temperature; avoid repeated temperature fluctuations

• Aliquoting strategy:

  • For antibodies in larger volumes, prepare single-use aliquots upon receipt

  • For smaller volume antibodies (such as 20μl sizes containing 0.1% BSA), aliquoting is generally unnecessary for -20°C storage

• Buffer considerations:

  • Most commercial RNF20 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Avoid introducing contaminants that may promote microbial growth or degradation

• Freeze-thaw management:

  • Minimize freeze-thaw cycles, which can damage antibody structure

  • Allow antibodies to thaw completely at 4°C before use

  • Return to -20°C promptly after use

• Working dilution handling:

  • Prepare working dilutions fresh before each experiment

  • Avoid storing diluted antibodies for extended periods

• Shelf-life awareness:

  • Most RNF20 antibodies remain stable for one year after shipment when properly stored

  • Document receipt date and track antibody age

  • Consider validation testing for older antibodies before use in critical experiments

How can RNF20 antibodies be utilized to investigate its role in RNA Polymerase II promoter-proximal pausing?

Recent research has revealed RNF20's crucial role in RNA Polymerase II (Pol II) promoter-proximal pausing, particularly in endothelial cells . To investigate this function:

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Use RNF20 antibodies for ChIP-seq to map genome-wide binding patterns

  • Compare with Pol II occupancy, particularly focusing on pause sites

  • Include analysis of H2BK120ub1 distribution to correlate with RNF20 binding

• Proximity ligation assays:

  • Employ RNF20 antibodies in combination with Pol II antibodies to detect physical interactions

  • Compare interaction frequencies at highly paused versus non-paused genes

• RNF20 knockdown/knockout studies:

  • Use RNF20 antibodies to validate depletion efficiency

  • Combine with Pol II ChIP-seq or PRO-seq to measure pausing index changes

  • Focus analysis on VEGFA signaling genes, stress response genes, and cell cycle control genes identified as highly paused in endothelial cells

• Mechanistic investigations:

  • Use co-immunoprecipitation with RNF20 antibodies to identify interactions with ERG transcription factor

  • Examine how these interactions restrict ERG-dependent Pol II pause release

  • Investigate changes in mRNA processing of key angiogenesis regulators following RNF20 depletion

This multi-faceted approach can provide mechanistic insights into how RNF20-mediated histone modifications influence transcriptional pausing and subsequent gene expression programs critical for vascular development.

What is the significance of RNF20 in VEGFA signaling and angiogenesis, and how can researchers investigate this connection?

Recent studies have uncovered a critical role for RNF20 in regulating VEGFA signaling and angiogenesis . Researchers can investigate this connection through:

• Vascular cell models:

  • Use RNF20 antibodies to characterize expression patterns in different endothelial cell types

  • Compare expression levels between tip cells and stalk cells during angiogenic sprouting

  • Correlate RNF20 levels with VEGFA receptor expression and activation

• Alternative splicing analysis:

  • After RNF20 knockdown/knockout, examine changes in VEGFA isoform distribution

  • Use RNF20 antibodies in RNA immunoprecipitation (RIP) assays to investigate direct involvement in mRNA processing

  • Correlate H2BK120ub1 levels at the VEGFA locus with splicing patterns

• Notch signaling intersection:

  • Investigate RNF20 binding to Notch1 using co-immunoprecipitation with RNF20 antibodies

  • Examine H2B monoubiquitination at Notch target genes through sequential ChIP approaches

  • Analyze changes in Notch-dependent gene expression following RNF20 manipulation

• In vivo angiogenesis models:

  • Use RNF20 antibodies for immunohistochemistry to visualize expression during vascular development

  • In Rnf20-deficient models, examine tip cell specification and sprouting angiogenesis

  • Correlate vascular phenotypes with changes in VEGF-Notch signaling balance

These approaches can help elucidate the complex role of RNF20 in orchestrating the transcriptional and post-transcriptional events that regulate angiogenesis, potentially identifying new targets for therapeutic modulation of blood vessel formation .

How can researchers use RNF20 antibodies to investigate its potential role in cancer progression?

RNF20's functions in transcriptional regulation, histone modification, and involvement in p53 pathways suggest important roles in cancer biology. Researchers can employ RNF20 antibodies to explore these connections through:

• Expression profiling:

  • Use immunohistochemistry with RNF20 antibodies to analyze expression patterns across cancer types and stages

  • Compare expression between matched tumor and normal adjacent tissues

  • Correlate expression levels with patient outcomes and treatment responses

• Functional analysis in cancer models:

  • Validate RNF20 knockdown/knockout efficiency in cancer cell lines using Western blot

  • Examine changes in global H2BK120ub1 levels and distribution at oncogenes and tumor suppressor loci

  • Investigate MDM2 promoter occupancy and p53 pathway regulation through ChIP approaches

• DNA damage response studies:

  • Use immunofluorescence with RNF20 antibodies to track localization after induction of DNA damage

  • Investigate recruitment to DNA damage sites and colocalization with repair factors

  • Examine how RNF20 manipulation affects cellular sensitivity to genotoxic therapies

• Polyubiquitination target identification:

  • Use RNF20 antibodies for immunoprecipitation followed by mass spectrometry to identify novel cancer-relevant substrates

  • Specifically examine interactions with PA2G4 (EBP1) isoform 2, which has been identified as an RNF20 polyubiquitination target in cancer cells

  • Validate identified targets through in vitro ubiquitination assays

• Therapy response markers:

  • Evaluate changes in RNF20 expression or localization following treatment with epigenetic modulators or conventional chemotherapies

  • Determine if RNF20 expression levels correlate with treatment resistance mechanisms

This research direction could potentially identify RNF20 as a biomarker or therapeutic target in specific cancer contexts, particularly those involving dysregulation of epigenetic processes or DNA damage repair pathways.

How do monoclonal versus polyclonal RNF20 antibodies compare in research applications?

Researchers have access to both monoclonal and polyclonal RNF20 antibodies, each with distinct advantages for specific applications:

For critical experiments, researchers should consider validation testing both monoclonal and polyclonal antibodies to identify the optimal reagent for their specific experimental system and question. When studying RNF20 in complex with other proteins, epitope accessibility becomes particularly important, and polyclonal antibodies may offer advantages for capturing these interactions.

What controls should be included when validating RNF20 antibody specificity for a new experimental system?

Rigorous validation of RNF20 antibody specificity is essential for reliable experimental outcomes. A comprehensive validation approach should include:

• Positive controls:

  • Cell lines with confirmed RNF20 expression (HeLa, MCF-7, Jurkat)

  • Tissue samples with known RNF20 expression (mouse kidney, liver)

  • Recombinant RNF20 protein (full-length or epitope-containing fragment)

• Negative controls:

  • RNF20 knockdown or knockout samples (siRNA, shRNA, or CRISPR-mediated)

  • Immunodepleted samples where RNF20 has been pre-cleared

  • Irrelevant isotype-matched antibodies

• Cross-reactivity assessment:

  • Testing in multiple species if cross-species reactivity is claimed

  • Evaluation of potential cross-reactivity with RNF40 (unless specifically engineered not to cross-react)

  • Testing in cell types with varying RNF20 expression levels

• Multiple detection methods:

  • Independent confirmation using antibodies targeting different epitopes

  • Correlation of protein detection with mRNA expression data

  • Mass spectrometry validation of immunoprecipitated proteins

• Application-specific controls:

  • For IHC/IF: Peptide competition assays to confirm binding specificity

  • For ChIP: Comparison with published RNF20 binding profiles

  • For Western blot: Molecular weight confirmation and detection of expected changes after treatment

This systematic validation approach ensures that experimental results can be confidently attributed to authentic RNF20 detection rather than non-specific interactions or technical artifacts.