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Synthetic peptide conjugated to KLH derived from within residues 50 - 150 of Human PHD3/prolyl hydroxylase.
(Peptide available as ab30781.)
Our Abpromise guarantee covers the use of ab30782 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 5 µg/ml.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 27 kDa (predicted molecular weight: 27 kDa).|
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab30782 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.
ab30782 immunoprecipitating PHD3 from rat PC12 (whole cell lysate - the cells were either normoxia (21% oxygen) or hypoxia (1% hypoxia). A protein A matrix was used with the antibody at a dilution of 1/1000. A non-Abcam antibody was used in the subsequent western blot. The doublet observed is specific to PHD3 - most probably a modified form or an alternately spliced form. Note: This antibody IP's a hypoxia inducible species of ~27 kDa and ~30 kDa. This is the same molecular weight as the human and mouse isoform, not the SM-20 variant with the N-terminal mitochondrial leader sequence.
Lane 1: normoxia - input
Lane 2: hypoxia - input
Lane 3: normoxia - control antibody IP
Lane 4: hypoxia - control antibody IP
Lane 5: normoxia - ab30782 IP
Lane 6: hypoxia - ab30782 IP
ICC/IF image of ab30782 stained HepG2 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 ab30782 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 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.
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