Anti-DYKDDDDK (binds to Sigma FLAG®) (polyclonal)

Recombinant FLAG-protein was expressed in bacteria and purified with affinity chromatography. 1.5 µg of FLAG protein was used per in vitro SUMOylation assay in two different conditions: line A. in 30°C for 3 hours and in B: 30°C overnight. FLAG-protein-SUMO1 FLAG-protein Samples were separated on 10% SDS-PAGE and transferred to PVDP membrane by semidry blotting. Membrane was blocked with 5% skim milk in PBST (0.01% Tween 20) for 1h in RT with agitation. Then blot was incubated with primary antibody anti FLAG at dilution of 1:5000 in 5% skim milk/PBST for 1h in RT with agitation. After washes (3 times for 5 min. in PBST) blot was incubated with secondary antibody (anti rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) at dilution 1:25 000 for 1h in RT also with 5% skim milk. The blot was washed 3 times for 5 min. with PBST and developed for 2 min with ECL according to the manufacturer's instructions. Exposure time was 1.5 min.

Dr Agata Cieśla, Ludwików's lab, Laboratory of Biotechnology UAM, Poland