By H. Stewart Hendrickson (auth.), Mark Doolittle, Karen Reue (eds.)
Lipase and Phospholipase Protocols is a crucial new compilation of state-of-the-art innovations and techniques for the characterization, purification, and expression of mammalian lipases and phospholipases. The equipment contain the coaching of a number of radiolabeled and fluorescent substrates for the choice of lipolytic job, confirmed ideas for the expression and purification of recombinant lipases and phospholipases, and the characterization of lipases by way of such innovations as immunodetection, subunit dimension decision, lipase kinetics, and ligand interactions. additionally, there are ways for the molecular and genetic research of the lipases, the quantification of mRNA degrees, and in vitro-coupled transcription/translation. The protocols hide greater than a dozen particular enzymes, and lots of should be utilized on to all lipases and phospholipases.
Lipase and Phospholipase Protocols constitutes a hugely invaluable single-volume instruction manual of uncomplicated systems and techniques to enquire this designated category of enzymes. Its step by step structure works completely on the laboratory bench or by means of the pc, supplying functional details and examples that might result in profitable layout and execution of particular concepts. It not just furnishes a technical framework for the research of recognized lipases and phospholipases, yet presents well-established tools effectively daptable to the invention of recent lipase enzymes.
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Extra resources for Lipase and Phospholipase Protocols
Example text
16 Remove any precipitate by passing through a small (2 5 Y 2 cm) Sephadex G-25 column equlhbrated with 25 mM HEPES buffer, pH 8 0 17 Load the clear solution onto a HlLoad 16110 S Sepharose column attached to a FPLC system 18 Elute the refolded protein with a linear gradient of NaCl from 0 to 0 5 A4 m 25 mM HEPES buffer, pH 8 0 (see Note 2) 19 Collect the major protein peak eluted with approx 0 25 MNaCl, dialyze against water, and lyophlhze The lyophlllzed hp-PLA, can be stored mdefimtely at -20°C without any detectable loss of activity.
2. Nuorimetric Assay Unless otherwise specified, all chemicals are obtained from from Sigma (St. LOUIS, MO) and stored at room temperature. 72 mg/mL solutton m chloroform Trtethanolamme stored at room temperature Just before use, make 1 mL of a 32% solutton m methanol 0 02 MMOPS (3-[N-morpholmo] propane sulfomc actd) buffer, pH 7 4, contalnmg 5% bovme serum albumm, store at 4°C 0 02 A4 MOPS buffer pH 7 4, contammg 1% Trtton X- 100, store at 4°C 0 02 A4 MOPS buffer, pH 7 4, contammg 0 004 M EGTA Branson Somfier cell dtsruptor 185 UV ltght Perkm-Elmer L55 spectrofluortmeter.
Thus, from the contmuous mcrease m fluorescence mtenstty, substrate hydrolysts can be monitored as a measure of ltpase activity, using a calibratton curve obtamed by plotting fluorescence mtenstties vs concentratton of an unquenched standard (pyrenedecanotc acid) (see Figs. 2 and 3) Under condtttons of nonltmttmg substrate concentrattons whtch depend on the reaction medium, enzyme actrvrty IS lmearly proportional to enzyme concentratton. The detetectton limit for pure lipases IS >5 ng depending on the lipase type, the stabthty of the hpase, and the quality of the enzyme preparation.