By Mark Doolittle
A different choice of step by step equipment for the characterization, purification, and expression of mammalian lipases and phospholipases. The equipment comprise the guidance of quite a few distinctive substrates for the choice of lipolytic task, the isolation of purified lipase arrangements, and the improvement of excessive expressing recombinant structures. There also are equipment for the immunodectection and construction of immunological reagents and the research of biochemical houses, akin to subunit measurement, lipase kinetics, enzyme immobilization, and ligand interactions. The protocols covers greater than a dozen particular enzymes, and supply well-established equipment that may be tailored to the invention of recent lipase enzymes.
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Elute the bound protem with 1 A4 NaCl m the same buffer 11 Dialyze the eluted protein solution against 25 mA4 HEPES buffer, pH 8 0 12. Equthbrate a Mono S 5/5 column with 25 mM HEPES buffer, pH 8 0 13 Load the protein solutton onto the column, and elute with a linear gradient of NaCl from 0 to 2 A4 m the same buffer 14. Collect the major protein peak eluted with approx 0 8 M NaCI, dialyze against water, and lyophilize The lyophtltzed hs-PLA, can be stored indefinitely at -2O’C without any detectable loss of acttvtty 36 Cho et al.