Anti sheep irdye 680rd anti rabbit 800cw

Forty microlitre reactions were set up containing 40 mM Tris–HCl pH 7.5, 100 µM ATP (Adenosine triphosphate), 10 mM MgCl₂, and 2 µg of either BUB-1 (259–332) or H1. At t = 0, 15 ng/µl Cdk1/Cyclin B was added, before incubation at 30°C for 30 min. Aliquots were taken immediately after Cdk1/CyclinB addition at t = 0 and then again after the 30 min incubation. All samples were then incubated at 70°C for 15 min in a final concentration of 1× LDS buffer. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was then conducted on a NuPage 4–12% Bis–Tris gel (Thermo) with MES buffer before being stained with ProQ Diamond (Thermo) and imaged using Bio-Rad ChemiDoc. Once fluorescence was recorded, Coomassie staining was performed. For the western blot, SDS–PAGE was conducted as above with 67 ng of substrate protein per well before the western was conducted using a nitrocellulose membrane (GE Healthcare) and 1× NuPage transfer buffer (Thermo). The membrane was blocked using Intercept PBS blocking buffer (LI-COR), the primary antibodies used were anti-GST at 1:1000 (made in sheep), and anti-phospho Ser 283 1:20,000 (made in rabbit). Secondary antibodies were anti-sheep IRDye 680RD anti-rabbit 800CW (LI-COR), both at 1:50,000. The membrane was then imaged using LI-COR Odyssey CLx.