Rnr1 | Ribonucleoside-diphosphate reductase large subunit (Affinity Purified)

10 ul of total protein from  1 x 10⁸ cells of Saccharomyces cerevisiae, extracted with lysis buffer ( 20 mM Tris, pH 8, 50 mM Ammonium acetate, 2 mM EDTA, plus protease and phosphatase inhibitor cocktails) containing 10% TCA, and re-suspended and denatured in 1X Laemmli buffer for 10 min, were separated on 10% SDS-PAGE and blotted on to nitrocellulose membrane (0.45µm) for 1.5h at constant 0.5Ampere current using wet transfer. Blots were blocked (5% milk) for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1:5000 o/n at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horseradish peroxidase conjugated) diluted to 1:5000 in TBS-T for 1h at RT with agitation. The blot was washed as above and developed with Supersignal West pico chemiluminescent detection reagents (Thermo scientific). Exposure time was 1 minute.