The values for yield were calculated and present an approximate estimation of the real AX extraction yield, since not the whole extracted suspension (which amounts were at least 60 L), but only a portion of the isolates were dried due to the limited capacity of the freeze drier.
The extract suspension was centrifuged (12,000g, 4 °C, 1 min) to precipitate cellular debris and collect supernatant.
This volume increase improves the performance of the robot's liquid detection system, which is required in the next step when the affinity beads/cell extract – suspension is transferred into the filter plate.
To allow for efficient dispensing of the assay mixture into the 5 mm narrow bore NMR tube (Wilmad, Buena, NJ), the lysate or extract suspension and the substrates were mixed in an Eppendorf tube; diluting viscous cell lysates helped to place them into the tube.
The extracted cell suspension was cooled on ice for 3 min and the cell debris was removed by centrifugation at 5000 rpm for 5 min. The cell-free metabolite extract was again centrifuged at 14,000 rpm for 8 min. The supernatant was transferred into a 2 ml glass vial and dried in a vacuum centrifuge (CentriVap Concentrator System, Labconco, MO, USA).
The apparent concentration of the norspermidine spike in the sample was taken as the concentration of norspermidine in the 500 μl final volume of extracted pellicle suspension.
Protein was extracted via suspension in RIPA buffer (Boston Bioproducts) and heated to 95 °C for 10 min. SDS-PAGE was performed with 7.5% Mini-PROTEAN TGX precast gels (BioRad) at 100 V.
HEK293 cell lysates were extracted in suspension buffer (50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 10% glycerol, 1 m m EDTA, 0.8% Triton X-100) with added 'Complete' protease inhibitors (Roche) and Phosphatase inhibitor cocktail (Sigma).
MJM, GCC, SMS, FMMA collected the leaves and prepared the extract and suspensions.
Over the last two years, NASD has extracted fines and suspensions in 80 cases involving variable annuities at brokerage firms around the country.
