RNF40 Antibody

What applications are most reliable for RNF40 antibodies and what dilutions should be used?

RNF40 antibodies are primarily validated for Western Blotting (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), and Immunofluorescence/Immunocytochemistry (IF/ICC) applications. Based on multiple antibody validation data, the recommended dilutions vary by application:

For optimal results, titration experiments should be performed for each specific antibody and experimental system. Different antibody clones may require adjustment within these ranges .

What is the expected molecular weight of RNF40 protein in Western blot analyses?

The calculated molecular weight of RNF40 is 114 kDa based on amino acid sequence, but the observed molecular weight typically ranges between 110-140 kDa when detected by Western blotting . This variation may result from post-translational modifications, alternative splicing, or differences in gel systems. When validating a new RNF40 antibody, researchers should expect bands within this range and confirm specificity using appropriate controls such as RNF40 knockdown or knockout samples .

What cell lines and tissues are optimal for RNF40 antibody validation?

Several cell lines and tissues have been consistently validated for RNF40 antibody testing:

These models provide reliable positive controls for antibody validation. For knockout validation, CRISPR-mediated RNF40 knockout in HepG2 cells has been demonstrated to effectively eliminate RNF40 protein expression, confirming antibody specificity .

How can researchers distinguish between RNF40 and its paralog RNF20 in experimental settings?

Distinguishing between these paralogs requires careful antibody selection and experimental design:

• Antibody Selection: Use validated antibodies specifically tested for cross-reactivity. For example, RNF40 (D2R2O) Rabbit mAb has been confirmed not to cross-react with RNF20/BRE1A .

• Double Immunostaining: For co-localization studies, employ distinct host species antibodies (e.g., rabbit anti-RNF40 with mouse anti-RNF20) with fluorescent secondary antibodies of different wavelengths.

• siRNA Knockdown Controls: Include parallel knockdowns of RNF40 and RNF20 to verify antibody specificity. The following siRNA sequences have been validated for RNF40 knockdown:

  • AACGAGACCGAAGAGAAA

  • ACUCUGAGCUCCAAGAUAA

  • GAUCAAGGCCAACCAGAUU

  • GGAGGUCGUUCGUGAGACA

• IP-MS Verification: Immunoprecipitate with the antibody of interest followed by mass spectrometry to confirm target identity.

What strategies can be employed to study the interaction between RNF40 and its functional partners?

To investigate RNF40 interactions with partners such as RNF20, LIMA1, or other proteins:

• Co-Immunoprecipitation: Use anti-RNF40 antibody (dilution 1:50-1:100) for IP followed by immunoblotting for suspected interaction partners. For example, co-transfection of RNF40-Myc with LIMA1 mutants followed by anti-Myc immunoprecipitation effectively revealed their interaction dynamics .

• Proximity Ligation Assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity.

• ChIP-seq/ChIP-reChIP: For studying chromatin associations, RNF40 ChIP followed by sequencing can identify genomic binding sites. GSEA analysis of H2Bub1 and gene expression data has successfully identified RNF40-regulated gene networks .

• CRISPR-Cas9 Domain Mapping: Strategic deletion of specific domains can identify regions required for protein interactions.

• Ubiquitination Assays: To study RNF40's E3 ligase activity on partners like LIMA1, perform ubiquitination assays by immunoprecipitating the target protein after co-expression with RNF40 and ubiquitin, followed by anti-ubiquitin immunoblotting .

How can researchers effectively analyze RNF40-mediated histone modifications?

RNF40-mediated H2B monoubiquitination is critical for subsequent histone modifications. To study this cascade:

• ChIP-seq Analysis: Perform H2Bub1 ChIP-seq in control versus RNF40-depleted conditions. This approach revealed 3335 regions with significant changes in H3K27ac occupancy following Rnf40 deletion .

• Sequential ChIP: For analyzing co-occurrence of histone marks, perform ChIP for H2Bub1 followed by re-ChIP for H3K4me3 or H3K79me.

• Genome Browser Visualization: Integrate ChIP-seq data with RNA-seq to correlate changes in histone modifications with gene expression.

• Histone Inhibitor Studies: Compare RNF40 deletion with EZH2 inhibition to distinguish direct versus indirect effects, as demonstrated in studies of Hoxc gene regulation .

• Western Blot Analysis: Examine global levels of H2Bub1, H3K4me3, and H3K79me in RNF40-depleted cells, using histone extraction protocols optimized for preservation of histone modifications.

How do researchers reconcile contradictory findings about RNF40's role in transcriptional regulation?

RNF40 exhibits context-dependent functions in gene regulation:

• Cell Type-Specific Analysis: Perform parallel studies in different cell types. For example, RNF40 displays disparate roles in different breast cancer subtypes (oncogenic in PR-negative but tumor-suppressive in PR-positive) .

• Gene Set Enrichment Analysis (GSEA): This approach has revealed that RNF40 can both activate and repress genes. GSEA identified PRC2-suppressed genes being significantly enriched in Rnf40-null cells .

• Stage-Specific Examination: Assess RNF40 function at different developmental or disease stages. For example, RNF40 exhibits stage-dependent functions in osteoblast differentiation .

• Genetic Interaction Studies: Combine RNF40 manipulation with modulators of other pathways (e.g., PRC2 components) to disentangle complex regulatory networks.

• Genome-Wide Studies: Integrate RNA-seq with various ChIP-seq datasets (H2Bub1, H3K4me3, H3K27me3, H3K27ac) to map the regulatory landscape. This approach revealed that H2Bub1 loss affects both active and PRC2-repressed genes .

What experimental approaches can help determine whether RNF40 functions as a tumor suppressor or oncogene in specific cancer contexts?

RNF40 exhibits dual roles in cancer, necessitating careful experimental design:

• Cancer Subtype Stratification: Analyze RNF40 expression and patient survival data across cancer subtypes. In breast cancer, high RNF40 expression correlates with poor relapse-free survival specifically in PR-negative patients but not in all breast cancer patients .

• Functional Assays: Compare effects of RNF40 depletion on:

  • Proliferation (e.g., EdU incorporation)

  • Migration/invasion (transwell assays)

  • Apoptosis (caspase 3/7 activity)

  • Tumor formation (xenograft models)

• Pathway Analysis: Examine how RNF40 depletion affects key pathways:

  • In colorectal cancer, RNF40 loss decreased NF-κB target gene expression and increased apoptosis via caspase 3/7 activation .

  • In triple-negative breast cancer, RNF40 supports YAP1-mediated stem cell features by stimulating glycolysis .

• Therapeutic Response: Test whether RNF40 status affects response to conventional therapies or targeted agents.

• Metabolic Studies: Investigate RNF40's role in cancer metabolism, as it has been linked to lipid metabolism via LIMA1 and glycolysis in TNBC .

What methods are recommended for studying RNF40's role in DNA damage response (DDR)?

RNF40 is implicated in the DNA damage response through H2B monoubiquitination:

• DNA Damage Induction: Treat cells with DNA-damaging agents (ionizing radiation, etoposide, or doxorubicin) and monitor H2Bub1 dynamics and RNF40 recruitment.

• Chromatin Fraction Analysis: Separate chromatin fractions to monitor RNF40 recruitment to damaged DNA.

• DDR Factor Co-localization: Perform immunofluorescence co-staining of RNF40 with γH2AX, 53BP1, or BRCA1 at various time points after damage.

• HR/NHEJ Reporter Assays: Measure the efficiency of homologous recombination and non-homologous end joining in RNF40-proficient versus RNF40-deficient cells.

• Cell Cycle Checkpoint Analysis: Determine whether RNF40 depletion affects cell cycle checkpoints following DNA damage using flow cytometry.

• Genomic Instability Assessment: Measure chromosome aberrations, micronuclei formation, or sister chromatid exchanges in RNF40-depleted cells.

What are the optimal conditions for preserving RNF40 protein during experimental procedures?

RNF40 protein stability requires specific handling procedures:

• Storage Conditions: Store antibodies at -20°C, as they remain stable for one year after shipment. Aliquoting is generally unnecessary for -20°C storage .

• Lysis Buffer Composition: Use PBS with protease inhibitors, 0.02% sodium azide, and 50% glycerol at pH 7.3 to maintain stability .

• Proteasome Inhibition: When studying RNF40-mediated protein degradation, include MG132 (proteasome inhibitor) in experimental designs. This approach effectively restored LIMA1 expression levels in RNF40 overexpression studies .

• Protein Synthesis Inhibition: For protein stability studies, cycloheximide (CHX) chase assays can determine RNF40 targets' half-lives, as demonstrated with LIMA1 degradation kinetics .

• Sample Preparation: For Western blot applications, thorough denaturation in SDS loading buffer and complete reduction of disulfide bonds are essential for accurate detection of the 110-140 kDa RNF40 protein.

What controls are essential when using RNF40 antibodies in various applications?

Proper experimental controls for RNF40 antibody applications include:

• Positive Controls:

  • Cell lines: HEK-293, HeLa, A375, and HepG2 cells consistently show detectable RNF40 expression .

  • Tissues: Mouse liver and brain tissues show reliable RNF40 expression .

• Negative Controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG)

  • RNF40 knockdown using validated siRNAs or CRISPR-Cas9 systems with the following guide RNA sequences:

    • Control sgRNA: 5'-CACCGGCACTACCAGAGCTAACTCA-3'

    • RNF40 sgRNA1: 5'-CACCGCCAGGCAACAAACGCGCCGC-3'

• Specificity Controls:

  • Pre-absorption with immunizing peptide

  • Sequential dilution series to confirm signal specificity

  • Cross-validation with multiple antibody clones

• Technical Controls:

  • Loading controls (GAPDH, β-actin) for Western blotting

  • DAPI nuclear counterstain for immunofluorescence

  • Non-immune serum controls for immunohistochemistry

Implementing these controls ensures reliable and reproducible results when studying RNF40 expression and function in research applications.