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).