Flamingo fluorescent protein stain

Proteinase K (PK) from a frozen aliquot stored at -20°C was diluted to 200 μg/mL in either water or Solution 2 and stored at room temperature or 4°C for 1 to 19 days. As a control, fresh PK was diluted to 200 μg/mL and kept on ice for 15 minutes prior to setting up the reactions. Each reaction contained 4 μL of PK solution, 2 μL of 1 mg/mL BSA in water, and 14 μL of either water or Solution 2. BSA was added last, and reactions were incubated for 1 hour at room temperature. Reactions were stopped after 1 hour by adding 4 μL of SDS loading buffer (200 mM Tris pH 6.8, 400 mM DTT, 10% BME, 8% SDS, 0.4% bromophenol blue, 40% glycerol) and heating the tubes at 95°C for 2 minutes. 12 μL of each reaction was separated on a 4–20% SDS-PAGE gradient gel (Bio-Rad Laboratories, Hercules, California, USA). The gel was fixed and stained with Flamingo Fluorescent protein stain (Bio-Rad) following the manufacturer’s instructions and imaged on a Chemidoc (Bio-Rad) imaging system. Undigested BSA migrates at ~66 kDa, while digestion products migrate below ~30 kDa.

Reactions (20 μl) contained 500 nM His₆‐UBE1, 2 μM UBE2L3, 2 μM HOIL‐1, 10 μM Cy5‐ubiquitin (South Bay Bio) and 10 mg/ml bovine liver glycogen (Sigma) in phosphate buffered saline pH 7.4 containing 0.5 mM TCEP and 5 mM Mg²⁺‐ATP. Reactions were incubated at 37°C for 1 h with gentle mixing. Where indicated, reactions were then incubated in the presence of 50 μg/ml (~0.1 units) human salivary α‐amylase (Sigma) or 1.5 M hydroxylamine for a further 60 min at 37°C. Reactions were stopped by adding NuPAGE LDS sample loading buffer supplemented with 50 mM DTT, and denatured at room temperature for 10 min. Reaction products were separated by gel electrophoresis on 4–12% Bis–Tris gradient gels using MES SDS running buffer (Invitrogen) and stained with Flamingo fluorescent protein stain (Bio‐Rad). Visualisation of the fluorescent signals was performed using the ChemiDoc MP Imaging System (Bio‐Rad) and ImageJ software (Schneider et al, 2012 (link)). In the case of assays using glycogen as substrate, these gels were then stained with periodic acid‐Schiff stain using the Pierce Glycoprotein Staining Kit (Thermo Scientific) and Coomassie stained with InstantBlue Protein Stain (Expedeon).