Anti myc polyclonal antibody

Yeast two-hybrid (Y2H) assays were performed according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech). Each pair of indicated genes were cloned into pGBKT7-DEST for fusing with GAL4 DNA binding domain (BD) or pGADT7-DEST for fusing with GAL4 activation domain (AD). Yeast competent cells (Y2H Gold) was co-transformed with bait and prey constructs, followed by 10-fold serial dilution and plating onto synthetic defined (SD) yeast leucine and tryptophan dropout medium (SD/-Leu/-Trp) or leucine, tryptophan, histidine and adenine dropout medium (SD/-Leu/-Trp/-His/-Ade). The transformants were allowed by 4- to 6-day growth on the dropout mediums at 28°C. For immunoblot detection of protein expression in yeast, the co-transformed yeast cells were propagated in liquid SD/-Leu/-Trp medium, and the cultures were harvested at OD₆₀₀ of 0.5. The total proteins were extracted by using Yeast Protein Extraction Reagent (Takara), followed by immunoblot detection with anti-HA monoclonal or anti-Myc polyclonal antibody (Abcam). Membrane yeast two hybrid (MYTH) assays were exactly performed according to the user manual of DUALmembrane starter kits (Dualsystems Biotech). Yeast competent cells (NMY51) were co-transformed with each pair of the indicated constructs, and plated onto SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade mediums.