Purification of mediator from HeLa cell lines expressing a flag-tagged mediator subunit.
The Mediator (Med) complex plays a key role in promoter-specific activation of transcription by RNA polymerase II (Pol II). Med is a major target of activators and can be used in many types of affinity binding and immobilized template studies to evaluate interactions with individual activators. Additionally, all of the Med subunits have been cloned and can be subjected to individual structure-function analyses to learn how a specific activator interacts with a particular subunit. This protocol presents a simple affinity-based method to purify Med complex from HeLa cells stably expressing the Flag-tagged Intersex protein.
Carey
Michael F
MF
Peterson
Craig L
CL
Smale
Stephen T
ST
D002846:Chromatography, Affinity / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D056892:Mediator Complex / Q000302:isolation & purification*; D021122:Protein Subunits / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D012117:Resins, Synthetic
D056892:Mediator Complex; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012117:Resins, Synthetic