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Synthetic peptide conjugated to KLH derived from within residues 650 to the C-terminus of Human DNA polymerase eta.
(Peptide available as ab19077.)
Our Abpromise guarantee covers the use of ab17725 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/400 - 1/2000. Detects a band of approximately 79-85 kDa (predicted molecular weight: 79 kDa).|
|ICC/IF||Use at an assay dependent concentration. PubMed: 25421715|
ane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: DNA poly eta knockout HAP1 cell lysate (20 µg)
Lane 3: XP30RO cell lysate (20 µg)
Lane 4: MRC5 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab17725 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab17725 was shown to recognize DNA pol eta when DNA pol eta knockout samples were used, along with additional cross-reactive bands. Wild-type and DNA pol eta knockout samples were subjected to SDS-PAGE. ab17725 1ug/ml and ab8245 (loading control to GAPDH) at a dilution of 1/10 000 were incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
XP30RO and MRC5 cell lysates were provide and a gift from Prof Catherine Green (Wellcome Trust Centre for Human Genetics, Univeristy of Oxford)
60% confluent 6cm dish of each cell type was washed in PBS then scraped into 150µl of SDS-PAGE buffer, boiled for 5 minutes then sonicated for 10 minutes. 20µl of each sample was loaded in triplicate on a 6.5% polyacrylamide denaturing gel, then transferred to nitrocellulose. The membrane was blocked in 5% non-fat milk in PBS plus 0.1% Tween20 for 30 minutes. The primary antibody, ab17725, was incubated with the membrane in PBS plus 0.1% Tween20 and 5% milk overnight. Where the immunizing peptide was used, 5µl of the peptide was incubated with 5ul of antibody for ten minutes prior to dilution in 2ml of 5% milk in PBS plus 0.1% Tween20, which was then incubated with the blocked membrane overnight.
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