Pseudomonas aeruginosa PA3904 pull down coupled to LC-MS/MS

pETDUET His-PAAR4 and pETDUET Flag-PAAR4 were transformed into BL21 E. coli DE3 and expressed as described above.  An equal volume of PAO1 retS was grown to an OD600 of 2.0.  E. coli expressing His-PAAR4 or Flag-PAAR4 and Pseudomonas cells were harvested by centrifugation and lysed as described above.  Lysates of both species were combined and His-PAAR4 or Flag-PAAR4 were purified as before.  Eluted sample was mixed with SDS-loading buffer, boiled and run on a 12% SDS-PAGE for 0.6 cm.  After coomassie staining and de-staining, the resulting band was excised and used for LC-MS/MS.