Pierce coomassie plus bradford protein assay

Whole seedlings, root base cells, and root tips containing stem cells were recollected and grinded in liquid N₂. Protein extracts were obtained with native lysis buffer (300 mM NaCl, 100 mM Hepes pH 7.4, 2 mM EDTA, 2% Triton X‐100) supplemented with 1X plant protease inhibitor (Sigma). Cellular debris was removed with 80,000 g centrifugation for 10 min at 4°C. Supernatant was recollected and protein concentration determined with Pierce Coomassie Plus (Bradford) Protein‐Assay (Thermo Scientific). A cellulose acetate membrane filter (GE Healthcare Life Sciences) was placed in a slot blot apparatus (Bio‐Rad) coupled to a vacuum system. Membrane was equilibrated with 3 washes with equilibration buffer (native buffer supplemented with 0.5% SDS). 200 µg of protein extract was supplemented with SDS at a final concentration of 0.5% and loaded and filter through the membrane. Then, the membrane was washed with 0.2% SDS. Retained aggregates of actin were detected using anti‐actin antibody [1:5,000] (Agrisera, AS132640). Extracts were also analyzed by SDS‐PAGE to determine total actin levels.

Total protein extracts were obtained from Arabidopsis lyophilized powder. The powder was resuspended on ice‐cold TKMES homogenization buffer (100 mM Tricine‐potassium hydroxide pH 7.5, 10 mM KCl, 1 mM MgCl₂, 1 mM EDTA, and 10% [w/v] Sucrose) supplemented with 0.2% (v/v) Triton X‐100, 1mM DTT, 100 µg/ml PMSF, 3 µg/ml E64, and 1X plant protease inhibitor (Sigma). The resuspended sample was centrifuged at 10,000 ×g for 10 min at 4°C and the supernatant recovered for a second step of centrifugation. Protein concentration was determined using the kit Pierce Coomassie Plus (Bradford) Protein‐Assay (Thermo Scientific). Approximately 40–50 µg of total protein was separated by SDS–PAGE, transferred to nitrocellulose membrane, and subjected to immunoblotting. The following antibodies were used anti‐CCT7 [1:1,000] (Abcam, ab170861) and anti‐Actin [1:5000] (Agrisera, AS132640).