The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 0.1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Potent inhibitor of the complement membrane attack complex (MAC) action. Acts by binding to the C8 and/or C9 complements of the assembling MAC, thereby preventing incorporation of the multiple copies of C9 required for complete formation of the osmolytic pore. This inhibitor appears to be species-specific. Involved in signal transduction for T-cell activation complexed to a protein tyrosine kinase. The soluble form from urine retains its specific complement binding activity, but exhibits greatly reduced ability to inhibit MAC assembly on cell membranes.
Involvement in disease
Defects in CD59 are the cause of CD59 deficiency (CD59D) [MIM:612300].
Contains 1 UPAR/Ly6 domain.
N- and O-glycosylated. The N-glycosylation mainly consists of a family of biantennary complex-type structures with and without lactosamine extensions and outer arm fucose residues. Also significant amounts of triantennary complexes (22%). Variable sialylation also present in the Asn-43 oligosaccharide. The predominant O-glycans are mono-sialylated forms of the disaccharide, Gal-beta-1,3GalNAc, and their sites of attachment are probably on Thr-76 and Thr-77. The GPI-anchor of soluble urinary CD59 has no inositol-associated phospholipid, but is composed of seven different GPI-anchor variants of one or more monosaccharide units. Major variants contain sialic acid, mannose and glucosamine Sialic acid linked to an N-acetylhexosamine-galactose arm is present in two variants. Glycated. Glycation is found in diabetic subjects, but only at minimal levels in nondiabetic subjects. Glycated CD59 lacks MAC-inhibitory function and confers to vascular complications of diabetes.
Cell membrane. Secreted. Soluble form found in a number of tissues.
Surface antigen recognized by monoclonal antibody 16.3A5 antibody
T cell activating protein antibody
Western blot - Anti-CD59 antibody (ab69084)
All lanes : Anti-CD59 antibody (ab69084) at 1 µg/ml
Lane 1 : Wild-type HAP1 whole cell lysate Lane 2 : CD59 knockout HAP1 whole cell lysate
Lysates/proteins at 40 µg per lane.
Lanes 1 - 2: Merged signal (red and green). Green - ab69084 observed at 14 kDa. Red - loading control, ab9484, observed at 37 kDa.
ab69084 was shown to recognize CD59 in wild-type HAP1 cells as signal was lost at the expected MW in CD59 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CD59 knockout samples were subjected to SDS-PAGE. Ab69084 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
IHC image of CD59 staining in Human normal placenta 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 (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab69084, 0.5µg/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.
ICC/IF image of ab69084 stained JEG3 cells. The cells were 4% formaldehyde 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 ab69084 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 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.
Jeon H et al. Activation of the complement system in an osteosarcoma cell line promotes angiogenesis through enhanced production of growth factors. Sci Rep8:5415 (2018).
Read more (PubMed: 29615744) »
Jeon H et al. Extracellular vesicles from KSHV-infected endothelial cells activate the complement system. Oncotarget8:99841-99860 (2017).
Read more (PubMed: 29245944) »