Ammonia is then concentrated for easier use as fertilizer, and remaining liquid is water clean enough to be reused as drinking water for the cows or for crop irrigation.
Those fractions judged to be greater than 95% pure were pooled and concentrated for further use.
Fractions containing both FMO and the RCC complex determined from the shoulder at 807 and 835 nm were collected and concentrated for future use.
As necessary, the proteins were dissolved or dialysed and concentrated for crystallisation using 10 kDa cut-off centrifugal concentrators (Millipore, Massachusetts, USA) in the buffers listed in Table 2. Protein concentration was measured using the Bradford assay (Bio-Rad™, California, USA).
The eluant was collected and concentrated for further purification using gel filtration chromatography (Superdex-200 column, GE Healthcare) in buffer containing 20 mmol/L HEPES pH 7.5, 200 mmol/L NaCl, 2 mmol/L DTT on the FPLC system (GE Healthcare Life Sciences).
Purified proteins were concentrated for crystallization trials using the Amicon Ultra Centrifugal Filter Devices of a 3000 MWCO (Millipore Corporation, Billerica, MA, USA).
Protein fraction of the medium was concentrated for about 100-fold using an Amicon Ultra-15 Centrifugal Filter Unites (Millipore, Billerica, MA) with a cut-off of 3000 daltons.
Prothrombin complex concentrate for use in this study was furnished by CSL Behring GmbH, Marburg, Germany, the commercial supplier of that product and sponsor of the study.
Conditioned media (CM) was first concentrated for 30 min at 3000 rpm using Amicon Ulita-5 centrifugal filter device (Millipore, Billerica US) as described by the manufacturer.
The other four were soaked in the substrate solution (100 mM Tris HCl buffer, pH 8.0, containing 0.1 mM phenazine methosulfate, 0.1 mM nitrotetrazolium blue chloride, 10 mM NAD and 100 mM d-lactate or dl-lactate) separately with gentle shaking for 60 min. Cell extracts of Bacillus sp. 2 6, L. pentosus DSM 20314 and L. plantarum DSM 20205 were concentrated and used for the native PAGE.
