This antibody gave a positive signal in the following cell lysates:
HeLa Whole Cell; HT 1080 Whole Cell; Jurkat Whole Cell; HepG2 Whole Cell; HeLa Whole Cell - Irradiated (5Gy); HeLa Nuclear; JEG-3 Whole Cell; NIH 3T3 Whole Cell; STO Whole Cell; RAW 264.7 Whole Cell; Caco 2 Whole Cell.
This antibody gave a positive result in IHC in the following FFPE tissue: Human colon adenocarcinoma.
This antibody gave a positive result in Flow Cytometry in HT1080 cells.
Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use 1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
1/200. Use with methanol-fixed cells.
Use a concentration of 5 µg/ml. Detects a band of approximately 156 kDa (predicted molecular weight: 156 kDa).
Abcam recommends using 3% milk as a blocking agent for this product.
FunctionComponent of the MRN complex, which plays a central role in double-strand break (DSB) repair, DNA recombination, maintenance of telomere integrity and meiosis. The complex possesses single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity, which are provided by MRE11A. RAD50 may be required to bind DNA ends and hold them in close proximity. This could facilitate searches for short or long regions of sequence homology in the recombining DNA templates, and may also stimulate the activity of DNA ligases and/or restrict the nuclease activity of MRE11A to prevent nucleolytic degradation past a given point. The complex may also be required for DNA damage signaling via activation of the ATM kinase. In telomeres the MRN complex may modulate t-loop formation.
Tissue specificityExpressed at very low level in most tissues, except in testis where it is expressed at higher level. Expressed in fibroblasts.
Involvement in diseaseDefects in RAD50 are the cause of Nijmegen breakage syndrome-like disorder (NBSLD) [MIM:613078]; also called NBS-like disorder or RAD50 deficiency. NBSLD is a disorder similar to Nijmegen breakage syndrome and characterized by chromosomal instability, radiation sensitivity, microcephaly, growth retardation, short stature and bird-like face. Immunodeficiency is absent.
Sequence similaritiesBelongs to the SMC family. RAD50 subfamily. Contains 1 zinc-hook domain.
DomainThe zinc-hook, which separates the large intramolecular coiled coil regions, contains 2 Cys residues that coordinate one molecule of zinc with the help of the 2 Cys residues of the zinc-hook of another RAD50 molecule, thereby forming a V-shaped homodimer. The two heads of the homodimer, which constitute the ATP-binding domain, interact with the MRE11A homodimer.
Post-translational modificationsPhosphorylated upon DNA damage, probably by ATM or ATR.
Cellular localizationNucleus. Chromosome > telomere. Localizes to discrete nuclear foci after treatment with genotoxic agents.
IHC image of Rad50 staining in Human colon adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab87918, 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.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - Rad50 antibody (ab87918)
All lanes : Anti-Rad50 antibody [20B5] (ab87918) at 5 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HT 1080 (Human fibrosarcoma) Whole Cell Lysate Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 5 : HeLa Whole Cell Lysate - Irradiated (5Gy) Lane 6 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate Lane 7 : JEG-3 (Human placental choriocarcinoma cell line) Whole Cell Lysate Lane 8 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 9 : STO (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 10 : RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell Lysate Lane 11 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate
Lysates/proteins at 20 µg per lane.
Secondary Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 156 kDa Observed band size : 156 kDa Additional bands at : 30 kDa,80 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 20 minutesAbcam recommends using 3% milk as a blocking agent for this product.
Immunocytochemistry/ Immunofluorescence - Rad50 antibody [20B5] (ab87918)Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada
ab87918 (1/200) staining RAD50 in assynchronous HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please see Abreview.
Overlay histogram showing HT1080 cells stained with ab87918 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab87918, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
References for Anti-Rad50 antibody [20B5] (ab87918)
has not yet been referenced specifically in any publications.
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