Western blot assay
TBS-T buffer (0.05% Tween-20) was used for the western blot assay. SDD-AGE and SDS-PAGE gels were transferred onto a nitrocellulose membrane (PALL) with transfer buffer (25 mM Tris, pH 8.0, 200 mM glycine, 20% methanol). Then, the membrane was washed 3 times with TBS buffer and was blocked by 5% non-fat powdered milk in TBS-T (5% non-fat powered milk/TBS-T, 1 h at room temperature). The membrane was subsequently washed 3 times with TBS-T buffer and was probed with anti-Sup35 sequence-specific antibody (Santa Cruz, sc25915) (1:5000 dissolved in 5% non-fat powered milk/TBS-T) for 30 min at room temperature, which was followed by 3 washes with TBS-T, each for 5 min. A secondary antibody (1:8000) conjugated with HRP was then incubated on the membrane for 30 min at room temperature. After being washed 3 times with TBS-T, the membrane was incubated with ECL solution (Pierce, product #34095), and 1 min later, X-ray film imaging was conducted in the dark room.
Blue native polyacrylamide gel electrophoresis(BN-PAGE) [25], SDS-PAGE and protein identification by MALDI-TOF/TOF
The ingredients for the preparation of the BN-PAGE gel were the same as those used for SDS-PAGE, except that preparation of the BN-PAGE gel was conducted in the absence of SDS. The basic (anode) buffer contained 20 mM Tris and 250 mM glycine. In addition to the ingredients in the basic buffer, the cathode buffer contained an additional ingredient, 0.1% Coomassie brilliant blue G250. At the beginning of the electrophoresis, an 80 V was applied for 20 min. A 20 mA constant current was used throughout the rest of the process (for approximately 1.5 h). The gel was then directly immersed in the destaining solution (distilled water:ethanol:acetic acid = 5:4:1) for 30 min.
The samples for SDS-PAGE were boiled with 6 × SDS loading buffer (300 mM Tris pH 6.8, 60% glycerol, 12% SDS, 0.6% bromophenol blue). To eliminate disulfide bonds, reducing agent (10 mM DTT) was added prior to boiling. All the gel concentrations used for SDS-PAGE and BN-PAGE were 12%.
The dissociated bands of compound I were excised from SDS-PAGE, which were digested with trypsin and then identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The MS plus MS/MS search was performed using the MASCOT search engine v3.5 (Matrix Science Ltd., London).