You are to purify, in its functionally-active form, a highly soluble, homotetrameric protein from a crude extract of E. coli. The protein contains an NADP binding site and has a pI of 9, with a subunit molecular weight of 35,000 Da. For this protein, select the four best techniques from the list below and place them in the correct order for the most reasonable strategy to obtain homogeneous protein.
(A) Precipitation of proteins using high ionic strength (40%) ammonium sulfate, followed by a second precipitation with 60% (w/v) ammonium sulfate in a batchwise precipitation.
(B) SDS-PAGE for 0.01 mg total protein.
(C) Cation exchange with CM-Sepharose at pH 7.5.
(D) Anion exchange with DEAE-Sepharose at pH 7.5.
(E) Affinity chromatography with Cibacron Blue-Sepharose (binds to NAD/NADP binding sites).
(F) Gel filtration with Sephadex, cation exchange chromatography with CM-Sepharose.
(G) Non-denaturing gel electrophoresis for 0.01 mg total protein.