Rnf168 Antibodies

E3 ubiquitin-protein ligase RNF168 (Rnf168)

E3 ubiquitin-protein ligase required for accumulation of repair proteins to sites of DNA damage. Acts with UBE2N/UBC13 to amplify the RNF8-dependent histone ubiquitination.

Recruited to sites of DNA damage at double-strand breaks (DSBs) by binding to ubiquitinated histone H2A and H2AX and amplifies the RNF8- dependent H2A ubiquitination, promoting the creation of'Lys-63'- linked ubiquitin conjugates. This leads to focus ubiquitinated histones H2A and H2AX in DNA lesions to the threshold necessary for recruitment of TP53BP1 and BRCA1.

Gene Information

Mouse
Gene Name:	Rnf168
Uniprot:	Q80XJ2
Entrez:	70238

Family

Belongs to:
RNF168 family

Alternative Names

E3 ubiquitin-protein ligase RNF168; EC 6.3.2; EC 6.3.2.-; FLJ39749; ring finger protein 168FLJ35794; RNF168

Molecular Weight

Mass (kDA):
64.779 kDA

Genomic Context

Mouse
Location:	16|16 B3
Sequence:	16;

Diseases

• Malignant Neoplasms
• Genomic Instability
• Neoplasms
• Immunologic Deficiency Syndromes
• Ataxia Telangiectasia
• Malignant Neoplasm Of Breast
• Ataxia
• Cell Transformation, Neoplastic
• Telangiectasis
• Fanconi Anemia
• Premature Aging Syndrome
• Chromosome Breakage

• Cytogenetic Abnormality
• Anemia
• Nerve Degeneration
• Dna Repair-deficiency Disorders
• Learning Disorders
• Bone Neoplasms

Pathways

• Dna Repair
• Cell Cycle
• Localization
• Double-strand Break Repair
• Cell Division
• Chromatin Remodeling
• Conjugation
• Histone Ubiquitination
• Aging
• V(d)j Recombination
• Dna Damage Checkpoint
• Mitosis
• Dna Replication

• Response To Ionizing Radiation
• Cell Death
• Cell Cycle Checkpoint
• Gene Silencing
• Immune Response
• Spermatogenesis
• System Development

PTMs

• Phosphorylation
• Ubiquitination
• Sumoylation
• Cleavage

• Methylation
• Deamination
• Deacetylation
• Acetylation

For details, visit:
bosterbio.com/bosterbio-gene-info-cards/Rnf168

Best Uses Of The RNF168 Marker In Boster Bio Experiments

If you are scientist and are planning to make use of the Boster bio system to conduct experiments you must read through the optimization tips and guides that the company offers. These guides will assist scientists make informed decisions, optimize their experiments, and improve their results. Every scientist will encounter problems in their research. Even though proper controls can reduce the risk of errors but there is always a possibility that something could go wrong with your experiment. Troubleshooting guides can help you determine possible sources of error , and how to eliminate them.

Primary antibodies with high-affinity

We recently created high-affinity antibodies for the RNF168 marker. With this marker, we were able to identify RNF168-containing peptides found in the target proteins with high affinity. We also developed an analysis of the structure of the RNF169-UDM2-ubNCP-related complex using the Jasco J-815 CD spectrometer. We used a cuvette of 0.1 cm in length and averaged 3 scans that ranged from 190 to 240 numerics. This resulted in a steady rise in temperature from 10 degrees to 70 degrees Celsius. The protein concentrations ranged between 25 and 35 mM.

We first identified RNF168 as a protein by co-transfection of 293T cells with GFP or GST-RNF168 WT RF*. In immunoblots, also, we discovered a GFP-tagged H2AX, which was translocated by an anti-FLAG antigen. Anti-FLAG antibodies detect the higher-molecular-weight proteins, such as histone H2AX and FLAG.

In addition to the RNF168-UBNCP-UBNCP complex an interaction between ubiquitin and RNF169 is also described. The canonical binding area for ubiquitin allows RNF169 to interact with ubiquitin. This is how RNF169 Ubiquitin -UDM2 binds to ubiquitin. This is the reason for the creation of high-affinity primates that utilize the RNF168 marker.

RZPD purchased human full length RNF168cDNA, which has the highest affinity for RNF168-based antibodi. It was then cloned with pGEX6P2 (GE Healtcare), and FLAG-pcDNA3.1(Invitrogen). The resulting lysates then were subjected to immunoblot analysis with antibodies against Ub and GST.

The protein is believed to regulate the chromatin structure by binding histone H2A to ubiquitination. RNF168 is also regulated by unknown factors. The protein is found near the site of the lesion and participates in checkpoint signalling pathways that promote homologous recombination as well as non-homologous joining. This is the reason why antibodies that target RNF168 can be useful in studying DNA damage.

RNF168-based peptides can be used to detect Ubiquitin. As mentionedearlier, the RNF168 and UDM2 peptides are quite similar in structure and function, but RNF168 is more specific. RNF169 is also involved in the recruitment and activation of ubiquitin at nuclear sites. Further, the ubiquitin-binding RNF168-derived protein peptides are involved in regulating nuclear events and proteasomal degradation.

High-affinity primary antibodies using the RNR168 marker can also be used to determine the presence of RNF168 in a variety of cancer cells. In this study, we employed HeLa cells that is transgenic for the RNF168 protein and immunoreactive in different cancer tissues. The cells that had been tagged with RNF168 were stained with NorthernLights 557-conjugated anti-sheep IgG secondary antibody, and then counterstaining with DAPI (blue).

The interaction module consists of two regions within the RNF168 molecule which are the MIU2 and the LRM2 domains. Both regions are conserved. The MIU2 domain contains an arginaine in the upstream region that is essential for stable interactions with the RNF168-UBN–NCP. The LANA peptide is also present in the RNF168–UDM2 complex.

RNF168 is an RING-finger-like protein with an exclusive Ub ligase activity. It binds to the H2A Lys13/Lys15 region of DNA and promotes the ubiquitination of histones belonging to the H2A family. RNF168 increases the concentrations of ubiquitinated proteins. It can also alter the highly ordered structure of chromatin by facilitating the binding of downstream repair factors including BRCA1.

The MBP-RNF169 (UDM2) was discovered to attach to the H2AK13Cub based -NCP and catalysis-prepared NCPs in screening tests that were conducted prior to the screening. Additionally, the RNF169-UDM2 compound creation was optimized using the ratio 1:5 of H2AK13Cub to RNF169(UDM2). After that, the RNF169-UDM2-UDM2 complex was made using a higher final PEG-4000 concentration (9 percent in v/w). The MBP-RNF169(UDM2)-UbNCPs bound by the modified construct exhibited significantly better thermostability than H2AK13Cub-NCP-UDM2-UDM2.

RNF169-UDM2 structures are also structurally relevant. Analyzing the structure of the RNF169-UDM2-UDM2 compounds by replica-averaged MD simulations, we discovered that the RNF169-UDM2 complex can discriminate between ubiquitylated chromatin and monoubiquitylated H2AK13/K15 nucleosomes.

The high-affinity primary antibodies derived from RNF168 can be utilized for research. They are useful for cancer immunoassays. To facilitate analysis and quantification of the expression of antigens, they can be conjugated with reporter enzymes or fluorescent tags. You can purchase the antibodies in polyclonal or monoclonal forms. All of the antibodies are suitable for research purposes. These antibodies are extremely useful in diagnosing applications and are encouraged to be used by researchers.

In addition to the RNF168 marker high-affinity primary antibodies may be made using CT-1H-13C HSQC spectrums of 15N,13C wild-type RNF169(UDM2) with increasing amounts of unlabeled Ubiquitin. These data were collected at 11.7 T, 35degC. We previously reported the primary antibodies RNF168-tagged with the RNF168 UDM2 marker as the most effective treatment for cancer.

An additional antibody can be used to control any subsequent steps in the assay. After reconstitution, primary antibodies lyophilized should be kept at a temperature of 4oC for 2 to 4 hours. Pre-adsorption using tissue extracts can reduce cross-reactivty while membranes with the target protein cut-out will minimize background signals. After reconstitution secondary antibodies can be prepared with a second antibody to serve as a control.

As we have already mentioned the size of the target protein is the primary determinant of target-recognition by antibody. The epitope that is the primary one should dominate cross-reactivity. It is crucial to conduct the experiment at the lowest amount of dilution possible, based on the antibody's Reactivity. It is also possible to test the specificity by comparing the antibody with an unrelated antibody, as well as to control samples that do not contain any target protein. To further verify the specificity of antibodies, you can use peptide competition assay.

A combination of biochemistry, structural methods and site-directed mutagenesis was used to identify the most appropriate antibodies that target human antigens. For this, the RNF168 marker was selected. In order to create high-affinity antibodies researchers used the RNF168 marker. The electron cryomicroscopy confirmed their results. However, this technique is not yet widely available as a tool that can be used in clinical practice.