Anti gst antibody

Five micrograms of GST-tagged wild-type Coi12p (GST-Coi12p) or GST-tagged Coi12p lacking the TPR domain (corresponding to amino acids 382–546 of Coi12p; GST-Coi12p-ΔTPR) was mixed with 5 μg of His-tagged wild-type Hsp82 (His-Hsp82p) or His-tagged Hsp82 lacking the last five amino acids (His-Hsp82p-ΔC) in 22 μl of GST-PD buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Triton X-100). Two microliters of the mix was mixed with 18 μl of 1× SDS–PAGE buffer and used as the “input” samples. The remainder of the samples was incubated at 4°C for 1 h. In parallel, 50 μl of a glutathione–Sepharose 4B bead suspension (GE Healthcare) was incubated with GST-PD buffer containing 0.1% BSA at 4°C for 1 h, and then, the beads were washed twice with GST-PD buffer. The protein complex containing the GST-tag was affinity-purified by incubating with the beads at 4°C for 1 h, followed by washing three times with GST-PD buffer at 4°C for 10 min each. The purified proteins were eluted from the beads by incubating with 40 μl of 1× SDS–PAGE buffer at 95°C for 5 min (“GST pull-down” sample). Ten microliters of the input samples and GST pull-down samples was analyzed by Western blotting using an anti-GST antibody (BD Bioscience) and an anti-6xHis antibody (Penta-His antibody, 5-PRIME).

To assess the impact of compounds on cIAP1 and cIAP2 binding to RIP1, 700,000 HEK293T cells were seeded into 100 mm cell-culture plates and transfected the next day with 6 μg of plasmid DNA, myc-RIP1 in pRK5 vector. After 24 h, cells were lysed in NP-40 buffer (20 mM Tris-HCl [pH 7.5], 135 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol) supplemented with EDTA-free protease inhibitors (Roche) and 1 mM DTT. Lysates from 6 plates were pooled and divided into equal 300 μL aliquots, into which 7 μg of purified GST-fusion proteins (GST-cIAP1-BIR3 or GST-cIAP2-BIR3) were added in the presence of various concentrations of SMAC mimetic compounds. The tubes were rocked overnight at 4°C, and the next day 20 μL of glutathione-Sepharose beads (GE Healthcare) was added to each tube and the tubes were rocked for an additional 2 h at 4°C. The GST-fusion proteins were recovered by centrifugation at 2,300 g for 30 sec, then washed 3-times in NP-40 lysis buffer. Finally, 20 μL of 2X Laemmli buffer was added and the samples were boiled, then fractionated by SDS-PAGE, followed by immunoblotting using anti-myc antibody (Roche) for detection of myc-RIP1 and anti-GST antibody (BD Biosciences, La Jolla, CA) for detection of GST-fusion proteins.