Mouse monoclonal antibody against flag tag

Open reading frames of Mic60^S.c. with a C-terminal FLAG tag (Mic60^S.c.-FLAG), of Mic60^P.d.-FLAG with an N-terminal mitochondrial targeting signal from Mic60^S.c., and of Mic60^R.s.-FLAG with an N-terminal mitochondrial targeting signal from Mic60^S.c. were cloned into p413 MET25 vector under the control of the MET25 promotor. Δmic60 (Mat a, his3-Δ1 leu2Δ0 met15Δ0 ura3Δ0; fcj1::kanMX4; Alkhaja et al., 2012 (link)) were either transformed with the empty plasmid or with plasmids containing the respective construct using the lithium acetate method. The cells were cultured in selective media (0.67% yeast nitrogen base without amino acids, 0.07% His dropout complete supplement mixture lacking histidine [MP Biomedicals], and 2% glucose or 3% glycerol, pH 5.0) and grown at 30°C. For the growth test, the cells were spotted in serial dilutions on selective glycerol plates and were incubated at 18°C. Mitochondria from such cells were isolated by using Zymolyase treatment, Dounce homogenization, and subsequent differential centrifugation. Mic60^S.c., Mic60^R.s., and Mic60^P.d. expression and mitochondrial targeting were monitored by SDS-PAGE and subsequent Western blot analysis of isolated mitochondria using mouse monoclonal antibody against FLAG-tag (Sigma-Aldrich).