Abstract
The chapter describes preparation and characterization of recombinant protein phosphatase 1 (PP1). Various methods for preparation of PP1C in bacteria, and methods for preparation of PP1C in Sf9 cells using the baculovirus expression system are presented. The chapter discusses the preparation of phosphorylated and thiophosphorylated (dopamine- and cAMP-regulated phosphoprotein,Mr 32,000)DARPP-32. Native PP1C is purified from rabbit skeletal muscle, using sequential chromatography on DEAE-cellulose, heparin-Sepharose, phenyl-Sepharose, Sephacryl S-200 and Mono-Q resins in order to recombinant PP1C. DARPP-32 and inhibitor-2 are prepared from E. coli as described. GST-tagged spinophilin and GSTPNUTS are prepared from E. coli as described. The chapter discusses Dephosphorylation of p-nitrophenyl phosphate (pNPP) and tyrosine-phosphorylated myelin basic protein. The chapter describes two different procedures that are used to prepare recombinant PP1, using the baculovirus method. Several different affinity chromatography procedures are used that utilized the His6 and GST tags attached to recombinant PP1. The chapter discusses immunoblotting of recombinant PP1C.
Published Version
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