Coomassie blue

Each reaction (total volume, 10 μl) contained 1 μg recombinant protein, 0.25 mM cold ATP, 5 μCi γ³²P-ATP and 350 ng CDK1–cyclin B recombinant human protein (Thermo Fisher PV3292) in kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij-35) supplemented with PhosSTOP (Sigma-Aldrich) and Complete EDTA-free proteases inhibitors (Sigma-Aldrich). The samples were incubated at 30°C for 10 min. The reactions were stopped by adding 3× Laemmli sample buffer and boiling at 95°C for 5 min. The samples were then resolved on a 10% acrylamide gel. The gel was stained with Coomassie Blue, dried and exposed to a phosphoimager screen (GE Healthcare). The results were analysed with a Typhoon FLA 9500 (GE Healthcare).

CALB L, the lipase B of Candida antarctica (CALB, EC 3.1.1.3, 19.1 U/mg protein; 7.50 mg protein/ml) was kindly provided by Novozymes (Bagsvaerd, Denmark). HyperLadder I markers were purchased from Bioline (Randolph, MA, USA). Tween 20 and Tween 80 were obtained from Panreac (Barcelona, Spain). 3-Aminopropyltriethoxysilane (APTS), NaBH₄, FeCl₂, FeCl₃, dimethylsulfoxide (DMSO), sodium dodecyl sulphate (SDS), p-nitrophenyl acetate (pNPA), bovine serum albumin, and Triton X-100 were purchased form Sigma-Aldrich (St. Luis, MO, USA). Coomassie Blue was obtained from GE Healthcare (Uppsala, Sweden). All other chemicals were supplied by Merck (Darmstadt, Germany). Non-edible oils (unrefined soybean, jatropha and cameline) were obtained from Bunge Ibérica, S.A. (Zierbena, Spain), Jatropha Hispania, S.L. (Toledo, Spain) and Camelina Company (Madrid, Spain), respectively. Olive oil used as a control was purchased from Carbonell (Madrid, Spain) and waste frying oil was obtained from local restaurants.

Sephacryl S-200 HR and sodium hydrosulfite were obtained from Sigma-Aldrich chemicals Co., Sweden. The disposable PD-10 columns, IPG strips, DTT, Iodoacetamide, Acrylamide, Bis-Acrylamide, Glycine, Methanol, Glacial Acetic acid, β-mercaptoenthanol and Coomassie blue, were obtained from GE Health Care. HNE was obtained from Cayman Chemical Company. Dialysis tubing was sourced from Spectrum laboratories, Inc. (Rancho Domingues, CA, USA). OFFGEL starter kit was procured from Agilent Technologies. All chemicals were of reagent grade or greater purity.