The cofactors were eluted using a flow rate of 1 mL/min with 5 min of isocratic phosphate buffer, followed by a 25 min linear gradient to 50% methanol, and finally a 5 min linear gradient to 75% methanol.
One-half gram of tissue was homogenized using a Bead-Beater at maximum speed for 2 min in 500 ml of phosphate buffer (20 mM Na2HPO4, 10 mM NaCl, pH 6.9).
Samples were suspended in 15.0 mL of 0.10 mol/L phosphate buffer (pH 7.4) by vortexing for 5 min, followed by addition of 10.0 mL of phosphate buffer and vortexing for 5 min.
After incubation for 15 min at room temperature, 10 mL of phosphate buffer was added and then centrifuged at 1500 xg for 20 min. The pellet was collected and then resuspended in a final volume of 0.5 mL of phosphate buffer which was then used for inoculation onto 2% Ogawa medium.
Cells were harvested by centrifugation at 6000 × g for 15 min and washed with 20 mL of phosphate buffered saline.
Bacterial strains were grown to mid-log phase, and approximately 4 × 10 bacteria were pelleted at 3220 g for 10 min and resuspended in 1 mL of phosphate-buffered saline.
To prepare crude protein extracts, 5 g of 21 dpi root nodules were frozen in liquid nitrogen, ground with a mortar and pestle to a fine powder, and mixed for 10 min at 4°C in 50 ml of phosphate-buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3) containing 2% (w/v) polyvinyl-polypyrrolidone (PVPP).
Five grams (5 g) of each sample were agitated vigorously with 10 ml of phosphate buffered saline for 5 min and centrifuged at 7500 rpm for 4 min.
After 5 min, 400 μl of phosphate buffer (0.5 M, pH 7.2) was added, followed by 3 ml L19-SIP (approximately 1.5 mg) and 35 μl IODO-GEN solution (1 mg ml−1).
Detached cells were washed twice in 50 mL of phosphate-buffered saline and 3 min centrifugation at 250× g.
