Instantblue comassie

Proteins from GAL4-PCGFs purification were separated by SDS–PAGE, using 4%–12% NuPAGE Novex Bis–Tris gels (Invitrogen) and NuPAGE MES SDS running buffer (Invitrogen) and then stained with Coomassie Blue using InstantBlue Comassie (Expedeon). Bands from gel were cut and digested with trypsin (Promega) and incubated for 16 h at 37°C for protein digestion. Then, peptide extraction was carried out and the resulting peptides mixture were combined, reduced in volume in a vacuum concentrator, desalted and concentrated using StageTip (Proxeon Biosystems) columns, washed with 30 μL of 0,1% Formic acid (FA) and finally eluted with 40 μL of 80% MeCN in 0,1% FA. The samples were concentrated in vacuum concentrator (Eppendorf concentrator 5301) for 5 min and peptides were dissolved in 7 μL of 0,1% FA. Approximately 5 μL of purified peptide mixture were analyzed on a LC–ESI–MS-MS Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Full scan MS spectra were acquired in a range of m/z 300–1800.

Proteins from RING1B WT and RING1B I53S purifications were separated for 2 cm by SDS–PAGE, using 4%–12% NuPAGE Novex Bis–Tris gels (Invitrogen)and NuPAGE MES SDS running buffer (Invitrogen) and then stained with Coomassie Blue using InstantBlue Comassie (Expedeon). Single bands from gel were cut and digested with trypsin (Promega) and incubated overnight at 37°C for protein digestion. Then, peptide extraction was carried out and the resulting peptides mixture were combined, desalted and concentrated using StageTip (Proxeon Biosystems) columns, washed with 30mL of 0,1% Formic acid (FA) and finally eluted with 40μL of 80% MeCN in 0,1% FA. The samples were concentrated in vacuum concentrator (Eppendorf concentrator 5301) and peptides were dissolved in 7μL of 0,1% FA. Approximately 6 μL of purified peptide mixture were analyzed on a LC–ESI–MS-MS Q-Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific), using a gradient of 80 minutes with a flow of 250 nL/min. Full scan MS spectra were acquired in arange of m/z 300–1650.