RNF2 Antibody

What is RNF2 and what are its primary functions in cellular processes?

RNF2 (also known as Ring1B or Ring2) is a member of the Ring finger protein family that functions primarily as an E3 ubiquitin ligase responsible for monoubiquitination of histone H2A at lysine 119 (H2AK119ub) . As a core component of the Polycomb Repressive Complex 1 (PRC1), RNF2 plays essential roles in gene silencing through chromatin modification . Research has demonstrated RNF2's involvement in multiple signaling pathways, including TGFβ signaling via down-regulation of LTBP2 and Wnt/β-catenin signaling through TCF7L1 destabilization . Additionally, RNF2 is critical for proper development of the central and enteric nervous systems, influencing the migration and differentiation of neural precursor cells .

What applications are RNF2 antibodies most commonly used for in research?

RNF2 antibodies find application across numerous experimental techniques in epigenetic and cancer research. Based on published literature, these antibodies are validated for Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence/Immunocytochemistry (IF/ICC), Immunoprecipitation (IP), Co-Immunoprecipitation (CoIP), Chromatin Immunoprecipitation (ChIP), and ELISA applications . The versatility of RNF2 antibodies enables researchers to investigate protein expression levels, protein-protein interactions, chromatin binding patterns, and subcellular localization across human, mouse, and rat samples .

How do I select the appropriate RNF2 antibody for my specific experimental needs?

Selection should be guided by your experimental application, target species, and specific research questions. Consider the following criteria:

• Validated applications: Ensure the antibody has been validated for your intended technique (e.g., WB, IF, ChIP)

• Species reactivity: Verify reactivity with your experimental model (human, mouse, rat)

• Antibody type: Choose between polyclonal (broader epitope recognition) or monoclonal (higher specificity)

• Published validation: Prioritize antibodies with established performance in peer-reviewed literature

For instance, if investigating RNF2 binding to chromatin, select antibodies validated for ChIP applications. The RNF2 antibody (16031-1-AP) shows reactivity with human, mouse, and rat samples across multiple applications, making it suitable for diverse experimental needs .

What are the optimal conditions for using RNF2 antibodies in Western Blot experiments?

For optimal Western Blot results with RNF2 antibodies, follow these methodological guidelines:

When troubleshooting weak signals, consider concentrating the antibody (1:1000 dilution) and extending incubation time to overnight at 4°C . Remember that RNF2 expression varies with cell type and tissue, with notable expression in cancer cells like melanoma cell lines (501Mel, WM983B) .

How should I perform Chromatin Immunoprecipitation (ChIP) experiments with RNF2 antibodies?

For successful ChIP experiments with RNF2 antibodies, implement this methodological workflow:

• Cross-linking: Fix cells with 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions.

• Chromatin preparation: Lyse cells and sonicate chromatin to fragments of 200-500 bp.

• Immunoprecipitation: Incubate sonicated chromatin with 2-5 μg of RNF2 antibody overnight at 4°C.

• Washing and elution: Perform stringent washes to remove non-specific binding, then elute protein-DNA complexes.

• Reverse cross-linking: Incubate at 65°C overnight to release DNA.

• DNA purification: Purify DNA for subsequent analysis by qPCR or sequencing.

Research demonstrates RNF2 binds to specific genomic regions, with over 300 RNF2-responding sequences identified through ChIP cloning in HEK293 cells . When analyzing ChIP-seq data, focus on genes involved in proliferation pathways, nucleotide synthesis, transcription regulation, and signaling pathways (particularly TGFβ signaling) .

What is the recommended protocol for RNF2 antibody use in Immunofluorescence (IF) studies?

For high-quality immunofluorescence detection of RNF2:

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 for 10 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum in PBS for 30-60 minutes

  • Apply primary RNF2 antibody at dilution 1:50-1:500 and incubate overnight at 4°C

  • Wash 3x with PBS

  • Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

• Nuclear counterstaining and mounting:

  • Counterstain with DAPI to visualize nuclei

  • Mount with anti-fade mounting medium

HeLa cells serve as positive controls for IF/ICC applications . When studying RNF2 localization, expect primarily nuclear staining pattern consistent with its function in chromatin modification and gene regulation.

How can I improve specificity and reduce background in RNF2 antibody experiments?

To enhance specificity and minimize background:

• Antibody validation strategies:

  • Include positive controls (cell lines known to express RNF2 like K-562, HEK-293)

  • Incorporate negative controls (secondary antibody only; RNF2 knockdown samples)

  • Verify signal absence in RNF2 knockout cells, as demonstrated in MEFs from RNF2 knockout mice

• Background reduction techniques:

  • Extend blocking time (2 hours at room temperature)

  • Add 0.1-0.5% Tween-20 to wash buffers

  • Optimize primary antibody concentration (start with 1:2000 for WB)

  • Consider protein extraction methods that maximize nuclear protein yield

• Signal verification approaches:

  • Confirm expected molecular weight (38 kDa)

  • Verify signal reduction with siRNA/shRNA knockdown

  • Use multiple antibodies targeting different epitopes

Research demonstrates the importance of proper RNF2 antibody validation, with knockdown experiments in 501Mel and WM983B cells confirming specificity of ChIP signals .

What are the common pitfalls when using RNF2 antibodies, and how can they be addressed?

Common challenges include:

When encountering variable results, remember that RNF2 expression levels change during disease progression, as demonstrated in melanoma where expression increases from nevi to primary tumors to metastatic lesions .

How can RNF2 antibodies be used to investigate epigenetic regulation mechanisms?

RNF2 antibodies enable sophisticated analysis of epigenetic regulation through several advanced approaches:

• Sequential ChIP (Re-ChIP): Perform first round of ChIP with RNF2 antibody, followed by a second round with antibodies against other PRC1 components or histone marks to identify co-occupancy.

• ChIP-seq integration analysis: Combine RNF2 ChIP-seq with transcriptome profiling to identify direct gene targets. This approach revealed 363 genes directly regulated by RNF2, including LTBP2, a modulator of TGFβ signaling .

• Histone modification correlation: Analyze relationships between RNF2 binding and H2AK119ub levels. In 501Mel and WM983B cells, RNF2 knockdown led to loss of H2AK119ub signal and increased activating histone acetylation marks on target gene promoters like LTBP2 .

• Dynamics of RNF2 complex formation: Use RNF2 antibodies in immunoprecipitation followed by mass spectrometry to identify novel interaction partners. This technique identified WASH as an RNF2-interacting protein through yeast two-hybrid screening .

The catalytic activity of RNF2 is essential for its gene-silencing function, as demonstrated by the fact that catalytically inactive mutants (RNF2 R70C or RNF2 I53S) failed to downregulate target genes like LTBP2 .

What methodologies enable investigation of RNF2's role in cancer progression?

To investigate RNF2's role in cancer progression, researchers can employ multiple sophisticated approaches:

• Tissue microarray (TMA) analysis: Use RNF2 antibodies for immunohistochemical analysis of large patient cohorts. This approach demonstrated progressive increase in RNF2 expression from nevi to primary melanomas to metastatic lesions across 480 tissue cores from 170 patients .

• Functional genomics with RNF2 modulation:

  • Overexpression studies with wild-type vs. catalytically inactive RNF2 mutants

  • shRNA-mediated knockdown in cancer cell lines (e.g., 501Mel, WM983B)

  • CRISPR-Cas9 knockout in appropriate model systems

• In vivo tumorigenesis assays: Xenograft models with RNF2-modulated cells revealed that RNF2 overexpression significantly increases tumorigenic potential in melanoma models, while knockdown reduces tumor burden .

• Pathway analysis integration: Combine RNF2 ChIP-seq with gene expression analysis to identify affected pathways. This approach identified enrichment in proliferation pathways, nucleotide synthesis, and hypoxia pathways in RNF2-occupied genes with increased expression .

• Analysis of metastatic potential: Invasion assays with RNF2-modulated cells demonstrated that RNF2 promotes invasive capacity through TGFβ signaling activation via LTBP2 downregulation .

How can RNF2 antibodies be used to study developmental processes?

For developmental studies, RNF2 antibodies can be employed in several specialized applications:

• Whole-mount immunohistochemistry: Use optimized RNF2 antibody dilutions (1:50-1:500) to visualize expression patterns in developing embryos.

• Lineage-specific expression analysis: Combine RNF2 antibody staining with markers for specific cell lineages to track developmental dynamics.

• Conditional knockout model analysis: Generate conditional RNF2 knockout models using Cre-loxP systems. Embryonic fibroblasts (MEFs) can be prepared from E14.5 embryos and infected with lentivirus encoding Cre recombinase to generate RNF2-deficient cells for developmental studies .

• Developmental phenotyping: RNF2 mutant zebrafish embryos show heart edema and pectoral fin phenotypes after 48 hours post-fertilization (hpf) , while whole-mount in situ hybridization (WISH) can be used to detect RNF2 expression patterns at different developmental stages.

• Neural development assessment: RNF2 plays a crucial role in the development of the central and enteric nervous systems, influencing migration and differentiation of neural precursor cells .

How should researchers interpret changes in RNF2 binding patterns across different experimental conditions?

When analyzing changes in RNF2 binding patterns:

• Integrate multiple data types: Combine ChIP-seq data with gene expression analysis to correlate binding changes with transcriptional outcomes. In HMEL-BRAFV600E cells, this approach identified TGFβ signaling genes as key RNF2 targets .

• Analyze histone modification correlation: Examine relationships between RNF2 binding, H2AK119ub levels, and other histone marks. RNF2 knockdown in melanoma cells led to loss of H2AK119ub signal and increased activating histone acetylation marks on target gene promoters .

• Consider context-dependent effects: RNF2 binding may have different consequences depending on cellular context and partner proteins. RNF2 occupancy led to both upregulation and downregulation of different gene sets .

• Pathway enrichment analysis: Apply bioinformatic approaches to identify biological processes affected by RNF2 binding changes. RNF2-occupied genes with increased expression show enrichment in proliferation pathways, while those with decreased expression associate with regulation of transcription .

• Validate key targets: Confirm direct regulation of selected targets through targeted ChIP-qPCR and expression analysis. This approach confirmed LTBP2 as a direct RNF2 target, with RNF2 occupancy correlating with repression .

What controls are essential for proper interpretation of RNF2 antibody-based experiments?

For robust data interpretation, implement these critical controls:

Research demonstrates the importance of proper controls; studies comparing wild-type RNF2 with catalytically inactive mutants (RNF2 R70C, RNF2 I53S) revealed that gene repression depends on RNF2's enzymatic activity . Similarly, RNF2 knockdown experiments confirmed the specificity of observed phenotypes in tumor formation assays .