application example

5-day-old light-grownwild-type (Columbia) (1) and hpr1-1 null mutant (SALK_143584) Arabidopsis thaliana seedlings were ground with a pestle in a 1.5 mL tube on dry ice (about 12 seedlings or enough to give ~ 20 µL of tissue), and double volume (~ 40 µL) of NuPAGE 2x loading buffer (Invitrogen) was added. After centrifugation, 20 µL of the supernatant was transferred to a fresh tube with 2.1 µL 0.5 M DTT and boiled at 100 °C for 5 minutes. Samples were loaded onto a NuPAGE 10% Bis-Tris gel (Invitrogen) next to Cruz Markers (Santa Cruz Biotechnology). After electrophoresis, proteins were transferred for 30 minutes at 24 V to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech) using NuPAGE transfer buffer (Invitrogen). The blot was blocked for 1 h at 4 °C in 8% non-fat dry milk in TBS-T (blocking buffer), and incubated overnight with agitation at 4˚C with primary antibodies, 1:10 000 diluted in blocking buffer. The antibody solution was decanted and the blot was rinsed twice for 5 min each at 4 °C in 8% non-fat dry milk in TBS-T with agitation. The blot was incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology) diluted to 1:5 000 in blocking buffer for 5 h at 4 °C with agitation. The blot was washed three times, for 5 min each, with TBS-T and developed with LumiGLO reagent (Cell Signaling Technology) according to the manufacturer's instructions. Exposure time was 3 seconds.

Courtesy of Sarah Bukhart and Bonnie Bartel, Rice University, USA