Lanes 1-2 & 5-6 : Anti-DNA polymerase eta antibody (ab17725) at 1/400 dilution
Lanes 3-4 : Anti-DNA polymerase eta antibody (ab17725) at 1/2000 dilution

Lanes 1 & 3 & 5 : XP30RO cell lysate (cells lacking endogenous DNA polymerase eta)
Lanes 2 & 4 & 6 : MRC5 cell lysate

Secondary
All lanes : Goat anti-rabbit HRP at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Exposure time: 20 minutes

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 5µl 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.