Cab1001

Detection of tagged proteins used anti-TAP (Thermo Scientific, CAB1001), anti-myc and anti-HA antibodies (Roche) and was performed as previously described [16] (link), [45] (link). Protein concentrations were determined using Quick StartTM Bradford Protein Assay (BioRAD) [78] (link) and checked with anti-Pfk1 antibodies recognizing yeast Pfk1 (1:50,000, kindly provided by Dr. J. Heinisch) or anti-Cdc19 serum (1:10,000, kindly provided by Dr. J. Thorner) so as to ensure equivalent loadings. For detection of the Hrr25 kinase in total yeast extracts and in immune precipitates, a generic anti-Hrr25 antibody [64] (link) was used (1:10,000 dilution). Antibody cross-linking to Dynabeads M-270 Epoxy (Invitogen), preparation of protein extracts and immune precipitation were performed according to the manufacturer's instruction and as described previously [16] (link), [79] (link). In general, 1 µg of antibody coupled to Dynabeads was used per 1 mg total cell extract in B60 buffer.

Yeast cultures were grown in YP+2% galactose or YP+2% dextrose to express or suppress TAP–Tom5, respectively. Log phase cultures (30 OD₆₀₀ units) were collected by centrifugation and resuspended in 400 µl of lysis buffer (250 mM NaCl, 50 mM Tris-HCl pH 7.4, 50 mM NaF, 5 mM EDTA, 1 mM DTT, 1 mM AEBSF, and 0.1% NP-40). Cells were disrupted with glass beads for 5 min at 4°C. The resulting lysate was centrifuged at 16,100×g for 5 min, and a sample of the supernatant was collected. The supernatant was then incubated under rotation with 20 µl of prewashed GFP–Trap_A bead slurry (Chromotek) for 1 h at 4°C. The supernatant was removed and the beads were washed with lysis buffer (3×1 ml, then 3×500 µl with 5-min incubations). Beads were resuspended in 100 µl of sample buffer (50 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 4% 2-mercaptoethanol, and 0.02% bromophenol blue) and eluted by heating at 65°C for 20 min. The eluted protein was subjected to SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-TAP (1∶5,000 dilution, Pierce, CAB1001) or anti-GFP (1∶5,000 dilution, Roche, 11814460001) antibodies. Goat anti-rabbit IgG-HRP (1∶5,000 dilution, Bio-Rad, 172-1019) and goat anti-mouse IgG-HRP (1∶5,000 dilution, Bio-Rad, 172-1011) were used as secondary antibodies. Blots were imaged with either Supersignal West Pico or Femto ECL substrate (Thermo Scientific).