Samples were mixed with sample loading buffer (Licor 928-40004) supplemented with DTT and incubated at 95° for 5 minutes. SDS-PAGE was performed with pre-cast 4–12% gradient gels (ThermoFisher NW04127BOX) in MOPS (ThermoFisher B000102) according to the manufacturer’s instructions.
For Coomassie staining, gels were washed thoroughly in water, incubated with ReadyBlue Protein Gel Stain (Sigma RSB-1L) overnight, and destained in water. For western blots, proteins were transferred to nitrocellulose membranes by semi-dry transfer (Biorad 1704158) according to the manufacturer’s instructions. Membranes were rinsed in water and stained with Revert 700 Total Protein Stain according to the manufacturer’s instructions. The membranes were then rinsed in TBS, rocked with Everyblot Blocking Buffer (Biorad 12010020) at room temperature for > 30 minutes, and rocked with primary antibody overnight at 4°. The membranes were washed with TBST, and rocked with IR800CW-labeled secondary antibodies for 30–60 minutes, washed with TBST, and imaged on a Licor Odyssey CLx.
For Coomassie staining, gels were washed thoroughly in water, incubated with ReadyBlue Protein Gel Stain (Sigma RSB-1L) overnight, and destained in water. For western blots, proteins were transferred to nitrocellulose membranes by semi-dry transfer (Biorad 1704158) according to the manufacturer’s instructions. Membranes were rinsed in water and stained with Revert 700 Total Protein Stain according to the manufacturer’s instructions. The membranes were then rinsed in TBS, rocked with Everyblot Blocking Buffer (Biorad 12010020) at room temperature for > 30 minutes, and rocked with primary antibody overnight at 4°. The membranes were washed with TBST, and rocked with IR800CW-labeled secondary antibodies for 30–60 minutes, washed with TBST, and imaged on a Licor Odyssey CLx.