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Synthetic peptide conjugated to KLH derived from within residues 2050 - 2150 of Human DNA PKcs.
(Peptide available as ab20406.)
Our Abpromise guarantee covers the use of ab18192 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||1/100. PubMed: 18559621ab171870-Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.|
|IHC-P||Use at an assay dependent concentration.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 460 kDa (predicted molecular weight: 460 kDa).Can be blocked with Human DNA PKcs (phospho S2056) peptide (ab20406).|
|ChIP||Use at an assay dependent concentration. PubMed: 20714222|
|ELISA||Use at an assay dependent concentration.
Direct ELISA with serially diluted ab18192 (0.2-1000 ng x mL-1), bound to immobilised phospho or control peptides (1 µg x mL-1).
|ICC/IF||Use a concentration of 1 µg/ml.|
Serially diluted ab18192 was bound to immobilised DNA PKcs phospho peptide (2052 - 2062; P-S2056) or DNA PKcs control peptide (2052 - 2062; both at 1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG (ab97080; diluted 50000 times) and signal was developed with TMB substrate.
ICC/IF image of ab18192 stained HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab18192 at 1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
ab18192 specifically recognizes a band at ~460 kDa corresponding to DNA PKcs in HeLa cells that have been treated with ionizing radiation (lane 1). This band is not detected in untreated cells (lane 2). The activity of the antibody is quenched by the addition of the immunizing (modified) peptide, ab20406 (lanes 3) but not the unmodified peptide, ab20407 (lane 5).
For the ab13823 irradiated HeLa cell lysate, the 4 hour post-treatment extract was used.
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