Experiment 7:
Isolation of overexpressed b-galactosidase from E coli
Use the E coli cell pellet provided; suspend in the BugBuster reagent and add Benzonase. Incubate for 10 to 20 minutes and then centrifuge in the microfuge in the cold to remove cellular debris. Repeat centrifugation.
Experiment 8 :
Preparation of the Ni column for affinity chromatography
Begin preparation of the column while preparing the cell extract. This involves washing the resin; charging the resin with Ni; washing again with bind buffer.
Ni affinity chromatography
Perform the purification of the b-galactosidase on the Ni affinity column. The chromatography involves equilibrating the Ni charged resin (see above) with bind buffer; applying the sample; washing the column with bind buffer and wash buffer; then eluting the b-galactosidase with elute buffer.
Fractions, about 1 mL, each should be collected. Initially the b-galactosidase activity should be determined for the individual fractions by a spot-check. Those fractions having significant activity should be analyzed for b-galactosidase activity by the standard fixed time assay and for protein concentration by either the Bradford or spectrophotometric method. Be careful when using the spectrophotometric method due the background absorbance of the high concentration of imidazole used in the elution buffer.