The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Detects a band of approximately 52 kDa (predicted molecular weight: 46 kDa).
Catalyzes the post-translational formation of 4-hydroxyproline in hypoxia-inducible factor (HIF) alpha proteins. Hydroxylates HIF-1 alpha at 'Pro-402' and 'Pro-564', and HIF-2 alpha. Functions as a cellular oxygen sensor and, under normoxic conditions, targets HIF through the hydroxylation for proteasomal degradation via the von Hippel-Lindau ubiquitination complex.
According to PubMed:11056053, widely expressed with highest levels in skeletal muscle and heart, moderate levels in pancreas, brain (dopaminergic neurons of adult and fetal substantia nigra) and kidney, and lower levels in lung and liver. According to PubMed:12351678 widely expressed with highest levels in brain, kidney and adrenal gland. Expressed in cardiac myocytes, aortic endothelial cells and coronary artery smooth muscle.
Defects in EGLN1 are the cause of erythrocytosis familial type 3 (ECYT3) [MIM:609820]. ECYT3 is an autosomal dominant disorder characterized by increased serum red blood cell mass, elevated serum hemoglobin and hematocrit, and normal serum erythropoietin levels.
Western blot - Anti-PHD2 / prolyl hydroxylase antibody [mAbcam74796] (ab74796)
Predicted band size : 46 kDa
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: PHD2 / prolyl hydroxylase knockout HAP1 cell lysate (20 µg) Lane 3: HeLa cell lysate (20 µg) Lane 4: HepG2 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab74796 observed at 52 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab74796 detected the expected band for PHD2 in wild type cells and the band was not seen in knockout cells. Additional cross reactive bands were observed in the wild type and knockout cells. Wild-type and PHD2 / prolyl hydroxylase knockout samples were subjected to SDS-PAGE. ab74796 at a concentration of 5 µg/ml and 181602 (loading control to GAPDH) diluted to 1/10,000 were incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772 and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.
Western blot - PHD2 / prolyl hydroxylase antibody [mAbcam 74796] (ab74796)
All lanes : Anti-PHD2 / prolyl hydroxylase antibody [mAbcam74796] (ab74796) at 5 µg/ml
Lane 1 : Human skeletal muscle tissue lysate - total protein (ab29330) Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 4 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution Developed using the ECL technique
Exposure time : 30 secondsThe 52-kDa band observed is comparable to the band detected by other commercially available antibodies to human prolyl hydroxylase (PHD2). Based on the immunogen sequence, this antibody is predicted to cross react with both isoform 1 (46 kDa) and isoform 2 (44 kDa) of PHD2. We believe that the band observed around 44-kDa corresponds to isoform 2.