RNF168 Antibody

What is RNF168 and why is it important in DNA damage response research?

RNF168 is an E3 ubiquitin-protein ligase that plays a crucial role in DNA damage response pathways. It acts as a key mediator in the recruitment of repair proteins to sites of DNA damage, particularly at double-strand breaks (DSBs). RNF168 works with UBE2N/UBC13 to amplify RNF8-dependent histone ubiquitination, promoting the formation of 'Lys-63'-linked ubiquitin conjugates . This amplification is essential for concentrating ubiquitinated histones H2A and H2AX at DNA lesions to the threshold required for recruitment of critical repair factors like TP53BP1 and BRCA1 .

The significance of RNF168 in research extends beyond basic DNA repair mechanisms to conditions like RIDDLE syndrome, which has been linked to frameshift mutations resulting in loss-of-function truncations of RNF168 . Additionally, its involvement in class switch recombination in the immune system makes it relevant to immunological research. Understanding RNF168 function provides insights into fundamental genome maintenance mechanisms and pathological conditions associated with genomic instability.

What types of RNF168 antibodies are available and how do they differ?

Available RNF168 antibodies vary in several important characteristics:

When selecting an RNF168 antibody, researchers should consider the specific experimental application, target species, and epitope region based on their research question. For example, antibodies targeting the carboxy terminus versus the middle region may have different detection capabilities depending on protein conformation or post-translational modifications.

How should RNF168 antibodies be validated before use in critical experiments?

A comprehensive validation approach for RNF168 antibodies should include multiple complementary methods:

• Positive control samples: Test the antibody on cell lines known to express RNF168, such as HeLa, HepG2, Jurkat, or MCF7 cells .

• Negative controls and specificity assessment:

  • Use RNF168 knockout or knockdown samples where possible

  • Test on non-expressing tissues or cells

  • Include isotype controls in immunostaining experiments

• Cross-validation with multiple detection methods:

  • Western blot: Verify a specific band at the expected molecular weight (65-70 kDa)

  • Immunofluorescence: Confirm expected nuclear localization pattern

  • Immunoprecipitation: Validate ability to pull down the target protein

• Lot-to-lot consistency testing: When receiving a new lot, perform side-by-side comparison with previous lots using standardized samples and protocols.

For optimal results, researchers should document the validation process thoroughly and include validation controls in all critical experiments. This approach helps ensure experimental reproducibility and reliable interpretation of results involving RNF168.

What are the optimal conditions for Western blot detection of RNF168?

Based on validated protocols across multiple antibody sources, the following conditions are recommended for optimal Western blot detection of RNF168:

Expected result: A specific band at approximately 65-70 kDa, with potentially minor variations based on cell/tissue type and post-translational modifications .

For mouse brain tissue, researchers have successfully detected RNF168 using a concentration of 0.5 μg/mL with sheep anti-mouse RNF168 antibody followed by HRP-conjugated anti-sheep IgG secondary antibody under reducing conditions .

How should RNF168 antibodies be optimized for immunofluorescence studies?

For successful immunofluorescence detection of RNF168, consider the following methodological approach:

• Fixation and permeabilization:

  • Immersion fixation with 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilization with 0.1-0.5% Triton X-100 (5-10 minutes)

  • Note: Methanol fixation may be tested if PFA results are suboptimal

• Antibody concentrations and incubation:

  • Primary antibody: 5-10 μg/mL (for R&D Systems AF7217) or 1:400-1:1600 dilution (for Proteintech 21393-1-AP)

  • Incubation time: 3 hours at room temperature or overnight at 4°C

  • Secondary antibody: Fluorophore-conjugated (e.g., NorthernLights™ 557-conjugated Anti-Sheep IgG)

• Counterstaining and controls:

  • Nuclear counterstain with DAPI is essential as RNF168 shows primarily nuclear localization

  • Include no-primary-antibody controls to assess background

  • When possible, include RNF168-depleted cells as negative controls

• Expected pattern:

  • Under normal conditions: Diffuse nuclear staining with possible nucleolar exclusion

  • After DNA damage induction: Distinct nuclear foci formation

  • Some studies also report cytoplasmic staining in certain cell types

Successful staining has been documented in HeLa cells, RAW 264.7 mouse monocyte/macrophage cell line, and HepG2 cells , making these useful positive control cell lines for protocol optimization.

What experimental approaches can assess RNF168 recruitment to DNA damage sites?

Several methodological approaches can be employed to study RNF168 recruitment to DNA damage sites:

• Laser micro-irradiation coupled with live cell imaging:

  • Transfect cells with fluorescently-tagged RNF168 (e.g., RFP-RNF168)

  • Induce localized DNA damage using laser micro-irradiation

  • Monitor recruitment kinetics through time-lapse microscopy

  • This approach allows visualization of the temporal dynamics of RNF168 recruitment

• Immunofluorescence following DNA damage induction:

  • Treat cells with DNA-damaging agents (e.g., bleomycin, ionizing radiation)

  • Fix cells at different time points after treatment

  • Perform immunostaining with RNF168 antibodies

  • Quantify foci formation and co-localization with γH2AX or other DDR markers

  • This method allows assessment of endogenous RNF168 recruitment

• Chromatin immunoprecipitation (ChIP):

  • Induce site-specific DNA breaks using endonucleases (e.g., I-SceI)

  • Perform ChIP using RNF168 antibodies

  • Quantify enrichment at break sites by qPCR or sequencing

  • This approach provides quantitative data on RNF168 recruitment to specific genomic loci

• Proximity ligation assay (PLA):

  • Use antibodies against RNF168 and known interacting partners (e.g., γH2AX, MDC1)

  • Perform PLA following DNA damage

  • Quantify PLA signals as a measure of protein-protein interactions at damage sites

When analyzing RNF168 recruitment, researchers should consider factors such as cell cycle phase, chromatin state, and the presence of other DDR factors like RNF8, which acts upstream of RNF168 in the signaling cascade .

What are common issues in RNF168 antibody experiments and how can they be resolved?

For researchers working with RNF168 antibody #21393-1-AP, Proteintech provides specific protocols for Western blot, immunofluorescence, and immunoprecipitation applications that may help in troubleshooting these issues .

How should researchers interpret contradictory results when studying RNF168 function with antibodies?

When facing contradictory results in RNF168 studies, consider these methodological approaches:

• Evaluate antibody specificity and epitope differences:

  • Different antibodies may target distinct regions of RNF168

  • Post-translational modifications or protein interactions might mask specific epitopes

  • Solution: Use multiple antibodies targeting different regions of RNF168

• Consider cellular context variations:

  • RNF168 function may vary across cell types or conditions

  • RNF168 can be regulated by other factors such as RNF126

  • Solution: Perform experiments in multiple cell lines and verify with RNF168 knockdown/knockout controls

• Assess experimental condition differences:

  • DNA damage type, dose, and timing can affect RNF168 dynamics

  • Cell cycle phase influences DDR signaling

  • Solution: Standardize experimental conditions and include appropriate time-course analyses

• Address potential technical artifacts:

  • Overexpression systems may yield non-physiological results

  • For example, contradictory findings regarding RNF126 effects on reporter systems have been reported

  • Solution: Confirm findings using endogenous protein detection and complementary techniques

• Validate with orthogonal approaches:

  • Combine antibody-based methods with genetic approaches (CRISPR/Cas9, siRNA)

  • Use functional readouts (e.g., DNA repair efficiency, cell survival)

  • Solution: Implement a multi-method validation strategy

When reporting contradictory results, researchers should thoroughly document all experimental conditions, antibody details, and cell line information to facilitate replication and resolution of discrepancies by the scientific community.

How can RNF168 antibodies be used to study its role in disease models?

RNF168 antibodies can be strategically employed to investigate its involvement in various disease models:

• RIDDLE syndrome research:

  • Use Western blot with RNF168 antibodies to detect truncated proteins in patient-derived cells

  • Perform immunofluorescence to assess nuclear localization and foci formation capacity

  • Analyze different RNF168 frameshift mutations (c.397dupG/c.1323_1326delACCA, c.391C>T, R131X, c295delG) that cause RIDDLE syndrome

  • Methodological approach: Compare RNF168 expression, localization, and function between patient-derived and healthy control cells

• Cancer research:

  • Analyze RNF168 expression levels in tumor tissues vs. normal tissues

  • Investigate RNF168 function in HPV-positive cancers, where increased RNF168 levels have been observed

  • Study how HPV E7 protein interaction with RNF168 (between MIU1 and MIU2 domains) affects DNA repair pathway choice

  • Methodological approach: Use co-immunoprecipitation with RNF168 antibodies to identify cancer-specific interacting partners

• Neurodegenerative disease models:

  • Examine RNF168 expression and activity in brain tissues from different regions (cortex, thalamus/hypothalamus)

  • Investigate RNF168's role in preventing genome instability in post-mitotic neurons

  • Methodological approach: Combine immunohistochemistry with functional DNA repair assays in neuronal models

• Immune system disorders:

  • Analyze RNF168's role in class switch recombination via its involvement in DSB repair

  • Study RNF168 function in B-cell development and antibody diversity

  • Methodological approach: Use ChIP with RNF168 antibodies at immunoglobulin loci in B-cells

For all disease models, researchers should combine protein detection using antibodies with functional assays to establish causative relationships between RNF168 dysfunction and disease phenotypes.

What methodological approaches can assess the interaction between RNF168 and other DNA damage response proteins?

To study RNF168's interactions with other DDR proteins, researchers can employ these advanced methodological approaches:

• Co-immunoprecipitation (Co-IP) assays:

  • Use RNF168 antibodies to pull down protein complexes

  • Recommended protocol: 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

  • Analyze interactions before and after DNA damage induction

  • Detect known interactors like RNF8, UBC13, histones H2A/H2AX, and 53BP1

  • Example: RNF126 has been shown to directly interact with RNF168 through in vitro pulldown assays using GST-RNF126 and FLAG-RNF168 or His-RNF168

• Proximity-dependent labeling:

  • Fuse RNF168 to BioID or APEX2

  • Identify proteins in close proximity to RNF168 at DNA damage sites

  • Compare interactome changes in response to different DNA damaging agents

• FRET/BRET analysis:

  • Tag RNF168 and potential interacting partners with appropriate fluorophores

  • Measure interaction dynamics in living cells

  • Assess how mutations or inhibitors affect these interactions

• Domain mapping experiments:

  • Generate RNF168 truncation or point mutants

  • Determine which domains are required for specific protein interactions

  • Focus on key functional domains such as the RING domain, MIU1, and MIU2

  • Example: The HPV E7 protein interacts with RNF168 at the region between MIU1 and MIU2

• Competitive binding assays:

  • Study how RNF126 reduces the level of RNF168 binding to UBC13

  • Use purified recombinant proteins to determine binding affinities

  • Assess whether interactions are direct or require mediator proteins

  • Perform in vitro ubiquitination assays to test functional consequences

These methodological approaches should be combined with functional assays to understand how protein-protein interactions influence RNF168's E3 ligase activity and its role in the DNA damage response pathway.

How can CRISPR-based approaches complement RNF168 antibody studies?

CRISPR-based technologies offer powerful complementary approaches to traditional antibody-based studies of RNF168:

• Endogenous tagging:

  • Use CRISPR knock-in to add fluorescent or epitope tags to endogenous RNF168

  • Advantages: Avoids overexpression artifacts, maintains physiological regulation

  • Applications: Live cell imaging of endogenous RNF168 recruitment to DNA damage sites

  • Methodological considerations: Choose tags that don't interfere with RNF168 function; validate tagged protein functionality

• Domain-specific mutation:

  • Generate precise mutations in functional domains (RING finger, MIU domains)

  • Create cell lines mimicking patient mutations found in RIDDLE syndrome

  • Applications: Structure-function analysis at endogenous expression levels

  • Methodological approach: Compare antibody-detected localization patterns between wild-type and mutant RNF168

• Controlled degradation systems:

  • Integrate degron tags for rapid, reversible RNF168 depletion

  • Applications: Temporal analysis of RNF168 requirements at different stages of the DNA damage response

  • Advantages over RNAi: More rapid, complete, and reversible protein depletion

• CUT&RUN or CUT&Tag with RNF168 antibodies:

  • Combine CRISPR-generated DSBs at specific genomic loci with antibody-based chromatin profiling

  • Applications: Genome-wide mapping of RNF168 recruitment patterns following DNA damage

  • Methodological advantage: Higher resolution and lower background than conventional ChIP

• CRISPR screens with RNF168 readouts:

  • Use antibody-based detection of RNF168 foci or ubiquitination as phenotypic readouts

  • Applications: Identify novel regulators of RNF168 recruitment and function

  • Methodological approach: Combine genome-wide CRISPR screening with high-content imaging

When integrating CRISPR approaches with antibody-based methods, researchers should carefully validate that genetic modifications don't create artifacts in antibody recognition or protein function.

What are the latest methodological advances in studying RNF168's role in chromatin modification?

Recent technological advances have enabled more sophisticated analysis of RNF168's chromatin-modifying activities:

• Mass spectrometry-based ubiquitinomics:

  • Allows comprehensive identification of RNF168-dependent ubiquitination sites

  • Can distinguish between different ubiquitin chain types (K63 vs. K48)

  • Applications: Identify the complete spectrum of RNF168 substrates beyond H2A/H2AX

  • Methodological consideration: Combine with RNF168 antibody-based immunoprecipitation to enrich for RNF168-associated substrates

• ChIP-sequencing with RNF168 antibodies:

  • Maps genome-wide distribution of RNF168 before and after DNA damage

  • Applications: Identify preferential binding sites and chromatin context dependencies

  • Methodological advances: CUT&RUN or CUT&Tag provides higher resolution than traditional ChIP-seq

• Chromatin accessibility assays:

  • ATAC-seq or DNase-seq to assess how RNF168 activity affects chromatin structure

  • Applications: Understand how RNF168-mediated ubiquitination influences chromatin compaction

  • Methodological approach: Compare accessibility profiles in wild-type vs. RNF168-deficient cells

• Super-resolution microscopy:

  • Techniques like STORM, PALM, or live-cell SIM provide nanoscale resolution

  • Applications: Visualize RNF168 recruitment dynamics at individual DSB sites

  • Methodological consideration: Requires highly specific antibodies or fluorescently tagged proteins

• In vitro reconstitution systems:

  • Purified components to recapitulate RNF168-mediated chromatin modification

  • Applications: Biochemical dissection of RNF168 substrate specificity and activity

  • Methodological advantage: Allows precise control of reaction components and conditions

When implementing these advanced approaches, researchers should validate findings using multiple complementary methods and carefully consider the limitations of each technique, particularly regarding antibody specificity and potential artifacts from protein tags or expression levels.