Sonifier cell disruptor b 12

Algal pellets were resuspended in an extraction buffer [NaCl 150 mM, Triton X-100 0.1% (v/v), Tris-HCl 50 mM pH 7.5], sonicated at 3 Amp for 15 s (Sonifier Cell Disruptor B-12, Branson) and thoroughly vortexed for 20 min. Triglyceride concentration was determined by using the enzymatic assay kit of BioVision (product K622-100).

Algal pellets were resuspended in an ice-cold extraction buffer (NaCl 150 mM, Triton X-100 0.1% (v/v), EDTA 1 mM, DL-dithiothreitol (DTT) 25 mM, complete EDTA-free protease inhibitor cocktail tablets (Roche), Tris-HCl 50 mM pH 7.8) added with polyvinylpolypyrrolidone (PVPP, insoluble in water) 2.5% (w/v) to complex polyphenols. Proteins were extracted by sonicating at 6 Amp for 30 s on ice (Sonifier Cell Disruptor B-12, Branson), vortexing for 30 s at 4°C, and repeating the procedure twice more. Protein extracts were centrifuged at 3000 g for 3 min at 4°C to spin down PVPP. The supernatant was centrifuged again at 10,000 g for 3 min to spin down cellular debris, and was then filtered with a 0.22 μm cellulose acetate-membrane syringe filter. Proteins were further purified according to the phenol phase separation procedure described by Carpentier et al. (2005 (link)), and were finally solubilized in an appropriate volume of a DIGE labeling buffer (urea 7 M, thiourea 2 M, ASB-14 2% (w/v), EDTA 0.5 mM, DTT 10 mM, Tris-HCl 50 mM pH 8.5) so as to reach a concentration comprised between 5 and 10 mg.mL⁻¹.

In case of respiratory and photosynthetic measurements, 1 mL and 6 mL of algal suspension, respectively, were centrifuged at 10,000 g for 5 minutes. Algal pellets were invariably resuspended in 1 mL extraction buffer (NaCl 150 mM, EDTA 1 mM, Triton X-100 1%, Tris–HCl 50 mM pH 7.5) and 25 mg polyvinylpolypyrrolidone (PVPP, insoluble in aqueous solution) were further added to complex polyphenols which would otherwise interfere with protein assay. Samples were sonicated at 3 Amp for 30 s on ice (Sonifier Cell Disruptor B-12, Branson) and thoroughly vortexed for 5 min at 4°C. PVPP was span down by centrifuging at 10,000 g for 5 min and the supernatant was used for determination of protein concentration (mg.mL⁻¹) by a Reagent Compatible/Detergent Compatible protein assay kit (Bio-Rad) derived from the Lowry-Ciocalteu method (for details see the manufacturer’s instructions) [71 ].