Cells were concentrated by centrifugation for 1 min at 4°C and 4000 rpm and washed with phosphate buffered saline (PBS).
In contrast, sp7+ cells were concentrated near the amputation site.
Supernatants from baculovirus-infected High5 cells were concentrated on a stirred ultrafiltration cell (Amicon, Danvers, MA, USA) using YM-10 membranes (Millipore, Bedford, MA, USA), and buffer was exchanged into binding buffer (50 mM phosphate, 300 mM NaCl, and 10 mM imidazole, pH 7.4) on PD-10 desalting columns (GE Healthcare, Little Chalfont, UK).
Whole-cell lysates were collected with radioimmunoprecipitation assay buffer, and medium from cells was concentrated using a centrifugal filter unit (EMD Millipore, Billerica, MA, USA).
Fractions eluted with the glycine HCl buffer were concentrated by ultracentrifugation and analyzed using SDS-PAGE (Fig. 4a).
Before assay, cell media samples were concentrated 10 times using Microcon spin devices (YM3, Millipore, Billerica, MA, USA) and subsequently re-suspended in Bio-Plex (Bio-Rad) assay buffer.
Escherichia coli, S. aureus and Y. enterocolitica cells from exponential growth phase were concentrated after centrifugation at 13,000×g for 3 min, washed with 0.1 mol L−1 PBS and re-suspended in the same buffer.
Enzymes were concentrated, buffer-exchanged into 50 mM Na-HEPES (pH 7.4), and stored at −80 °C.
Eluates were concentrated and subjected to buffer exchange with phosphate buffer pH 7.4 using Amicon ultra 10,000 MWCO centrifugal filter devices (Millipore).
Eluted proteins were concentrated and elution buffer replaced using a 0.5 ml 10 K microcon (Millipore).
