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. 2011 Jul 8;39(19):8342–8354. doi: 10.1093/nar/gkr551

Figure 2.

Figure 2.

Composition of purified med7 ts and med8 ts mediators. (A) Western blot showing the stability of a mutant Mediator complex after the thermo-inactivation of the med7-163 mutant protein. Lane 1 shows wild-type Mediator purified by conventional means. Lanes 2–4 show the isolation of Mediator using a one step affinity purification under mild conditions, from whole cell extracts of a wild-type strain grown at 30°C, the med7-163 strain shifted to 34°C (for 45 min) or grown at 23°C. The strains used in Lanes 2–4 all had a Flag-tag on the chromosomal copy of Med18p(Srb5p). Mediator was depleted from extracts using α-Flag agarose, and the form shown on the blot was eluted from the resin with Flag peptide. Amounts loaded are normalized so as to give an equal signal using an antibody against the native Med18p(Srb5p). (B) Western blot showing the stability of a mutant Mediator complex after the thermo-inactivation of the med4-6 mutant protein. Lane 1 shows wild-type Mediator purified from a Med18p(Srb5p) flag-tagged wild-type strain grown at 30°C. Lanes 2 and 3 show Mediator purified from whole cell extracts the Med18p(Srb5p) flag-tagged med4-6 strain grown at 23°C or shifted to 34°C (for 45 min) using a one step affinity purification under mild conditions. Mediator was depleted from extracts using α-Flag agarose, and the form shown on the blot was eluted from the resin with Flag peptide. Amounts loaded are normalized so as to give an equal signal using an antibody against the native Med18p(Srb5p).