P-TABM-6289 - P-TABM-6289
Yeast strains and epitope tagging: Saccharomyces cerevisiae (S. cerevisiae) BY4741 (MATa his3?1 leu2?0 met15?0 ura3?0) strains containing C-terminally tandem affinity purification (TAP) tagged versions of target proteins were obtained from Open Biosystems. The Spt5?CTR strain lacking 15 C-terminal hexapeptide repeats, amino acids 931 to 1063, of Spt5 was generated as described. BY4741 untagged wild-type strain (Open Biosystems) was used for mock IP in ChIP-Chip experiments. All epitope-tagged strains were validated. First, gene-specific PCR was performed to confirm that the TAP tag was at the correct genomic position. Second, Western blotting with anti-TAP (PAP, Sigma) antibodies was performed to verify if the tagged protein of interest was properly expressed. Third, the growth of the various tagged strains compared to the non-tagged wild-type strain was monitored to rule out any influence of the epitope tag on yeast growth. This was done by serial dilutions of the various yeast strains on YPD plates at 30 C for two days.