The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 213 kDa (predicted molecular weight: 213 kDa).
Use a concentration of 1 µg/ml.
FunctionMay have a role in checkpoint signaling during mitosis. Enhances TP53-mediated transcriptional activation. Plays a role in the response to DNA damage.
Involvement in diseaseNote=A chromosomal aberration involving TP53BP1 is found in a form of myeloproliferative disorder chronic with eosinophilia. Translocation t(5;15)(q33;q22) with PDGFRB creating a TP53BP1-PDGFRB fusion protein.
Sequence similaritiesContains 2 BRCT domains.
Post-translational modificationsAsymmetrically dimethylated on Arg residues by PRMT1. Methylation is required for DNA binding. Phosphorylated at basal level in the absence of DNA damage. Hyper-phosphorylated in an ATM-dependent manner in response to DNA damage induced by ionizing radiation. Hyper-phosphorylated in an ATR-dependent manner in response to DNA damage induced by UV irradiation.
Cellular localizationNucleus. Chromosome > centromere > kinetochore. Associated with kinetochores. Both nuclear and cytoplasmic in some cells. Recruited to sites of DNA damage, such as double stand breaks. Methylation of histone H4 at 'Lys-20' is required for efficient localization to double strand breaks.
53BP1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to 53BP1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab87097. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 213kDa: 53BP1.
ICC/IF image of ab87097 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab87097, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
IHC image of 53BP1 staining in human normal cervix formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab87097, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
References for Anti-53BP1 antibody (ab87097)
This product has been referenced in:
Carvalho S et al. SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint. Elife3:e02482 (2014).
Read more (PubMed: 24843002) »