RPB2 | DNA-directed RNA polymerase II subunit 2

10-40 µg/well of total protein extracted from 7-d-old Arabidopsis thaliana seedlings (grown in continuous light at 22ºC) with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.25% Triton X-100, 2 mM PMSF and 1x protease inhibitor cocktail (Roche) and denatured with 1XLaemli buffer at 100°C for 5 min, were separated in duplicate on 7% SDS-PAGEs and blotted 1h 15 min to PVDF (0.2 um), using wet transfer. Blots were blocked with TBS-T/5% milk for 1h/RT with agitation. Blots were incubated in the primary antibodies at a dilution of 1:1000 (anti-NRPB2) ON/4°C in TBS-T/5% milk with agitation. The anti-NRPB2 antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (rabbit anti-chicken IgG horse radish peroxidase conjugated, AS10 1489) diluted to 1:1000 in for 1h/RT with agitation. The two blots were washed as above and developed for 5 min with chemiluminescent detection reagent, following manufacture's recommendations.  Exposure time was 10 seconds (ImageQuant 800, 3x3 bins; Amersham).

Courtesy of Alberto Palacios-Abella from Dr. David Alabadi lab, CSIC-UPV, Spain