Glutathione coated sepharose beads

To detect the interaction between Eaf1 and Yaf9 in vitro, GST-Eaf1 (150 μg) and MBP-Yaf9 (100 μg) were incubated at 4 °C for 2 h within 1 mL lysis buffer supplemented with 1 mM GTP and 1 mM DTT. To pull down Yaf9 and Yaf9 mutants, GST-Eaf1 was purified by GST affinity chromatography on glutathione-coated Sepharose beads (GE Healthcare) in the lysis buffer, as recommended by the manufacturer. To pull down Eaf1 and Eaf1 mutants, MBP-Yaf9 was purified by MBP affinity chromatography on glutathione-coated Sepharose beads (GE Healthcare) in the lysis buffer. The beads were washed three times (10 min each) with the same buffer and then analyzed by co-immunoprecipitation (Co-IP) or IB with anti-GST antibody (Sigma) or anti-MBP antibody (NEB). The pGEX-4T1 or pSJ8 vector was used as control.

The recombinant GST-EB1 was expressed in E. coli JM109 (DE3) (Merck Biosciences) and BL21 CodonPlus (DE3) (Stratagene, UK), as previously described (Kammerer et al., 1995 (link)) using an autoinduction protocol (as described by Studier, 2005 (link)). GST–EB1-C-T was purified using glutathione-coated Sepharose beads (GE Healthcare). Purification was performed under denaturing condition using 8 M urea as described in the pET system manual (Merck Biosciences). Removal of the GST tag by thrombin was performed as previously described (Kammerer et al., 1995 (link)). Determination of protein concentration was achieved by measuring the absorbance of tryptophan and tyrosine residues at 280 nm (Edelhoch, 1967 (link)).

COS cells were washed with PBS, trypsinised, centrifuged and lysed in 500 µl ice-cold lysis buffer (1% NP40, 50 mM Tris-HCl pH 7.4, 120 mM NaCl, 2.5 mM EGTA 10 mM MgCl, supplemented with 2× protease inhibitor cocktail solution). Lysates were passed through a 27G needle ten times, centrifuged in a pre-cooled centrifuge at 4°C for 15 min at 800 g. The supernatant was pre-cleared with glutathione-coated Sepharose beads (GE Healthcare) for 1 h at 4°C, followed by centrifugation at 2000 g and aspiration of the supernatant. glutathione-coated Sepharose beads were incubated with either GST–EB1 or GST alone (control). After pre-clearing, the GST–EB1 or GST-coated beads were washed three times in PBS containing 0.01% Tween 20 (PBS-T), and were resuspended in the pre-cleared lysate and rotated for an hour at 4°C. The samples were centrifuged at 2000 g. The beads were washed four times in PBS-T and the bound proteins were eluted from the beads in 2× SDS-loading buffer. The individual fractions were then separated by SDS-PAGE and analysed by immunoblotting, using an anti-GFP antibody (1∶1000, Roche).