Clontech matchmaker gold yeast two hybrid system

Y2H assays were performed to detect interactions between proteins using the Clontech Matchmaker Gold Yeast Two-Hybrid System (TaKaRa). The fusion plasmids pGADT7 (AD vector) and pGBKT7 (BD vector) were transformed into Y187 and Gold yeast competent cells, respectively. Y187 cells were cultured on selective plates with synthetically defined (SD) medium lacking leucine (SD/-Leu), whereas Gold cells were cultured on plates lacking tryptophan (SD/-Trp). After 48–96 h, yeast were grown on SD/-Leu and SD/-Trp medium and then hybridized in 2× yeast extract peptone dextrose-adenine (YPDA) medium and selected by a double dropout (SD/-Leu/-Trp) plate. Interactions between proteins were detected based on the ability of the hybridized yeast to grow on quadruple dropout (SD/-Ade/-His/-Leu/-Trp) medium supplemented with X-α-Gal and aureobasidin A (TaKaRa).

Yeast two-hybrid (Y2H) assays were performed to detect interactions between proteins. Briefly, using the Y2H system (Clontech Matchmaker Gold Yeast Two-Hybrid System; TaKaRa), the fusion protein expression plasmids pGADT7 (AD vector) and pGBKT7 (BD vector) were transformed into the Y187 and Gold yeast strains, respectively, according to the manufacturer’s instructions. Y187 cells were cultured on selective plates with synthetically defined (SD) medium lacking leucine (SD/-Leu), whereas Gold cells were cultured on SD plates lacking tryptophan (SD/-Trp). After 3–5 days, yeast strains able to grow on SD/-Leu and SD/-Trp medium were hybridized in 2× yeast extract peptone dextrose (YPDA) medium and selected on double drop-out (SD/-Leu/-Trp) medium. Interactions between proteins were detected based on the ability of the hybridized clones to grow on quadruple drop-out (SD/-Ade/-His/-Leu/-Trp) medium supplemented with X-α-Gal and aureobasidin A (TaKaRa).

The yeast two-hybrid system was carried out to study the interaction between CgVDAC2 and CgBak using Clontech Matchmaker Gold Yeast Two-Hybrid System (TaKaRa, Shiga, Japan). pGADT7-CgVDAC2 and pGBKT7-CgBak were transformed into the Y187 and Gold yeast strains, respectively. Y187 cells were cultured onto selective plates with synthetically defined medium (SD) lacking leucine (SD/-Leu), while Gold cells were cultured on SD plates lacking tryptophan (SD/-Trp). After 3 d, positive yeast strains on SD/-Leu and SD/-Trp were hybridized in 2X yeast extract peptone dextrose (YPDA) medium and selected on double drop-out SD/-Leu/-Trp medium. The interaction between CgVDAC2 and CgBak was detected by the hybridized clones growing on quadruple drop-out SD/-Ade/-His/-Leu/-Trp medium supplemented with X-α-Gal and Aureobasidin A (TaKaRa, Shiga, Japan).