Zonal sedimentation on a sucrose gradientSimultaneously load 6 µg of recHA3 by pipetting them (use a 10 µl tip connected to a pipettor) just below the top of a 5-25% sucrose gradient containing a 60% sucrose cushion.
{"action": "load", "output": "", "reagent": ["6 µg of recHA3"], "device": ["a pipettor"]}
Manually collect fractions of 250 µl by slow pipetting.
{"action": "collect", "output": "", "volume": ["250 µl"]}
Rinse membranes with water.
{"action": "rinse", "output": "", "reagent": ["membranes"]}
Wash membranes 3 times with 10 ml 0.5% Tween-20/1x PBS for 10 min at room temperature with vigorous shaking. #####%%%%% output "the protein standards" are used in line10
{"action": "wash", "output": "the protein standards", "volume": ["10 ml"], "reagent": ["0.5% Tween-20/1x PBS"], "time": ["10 min"], "temperature": ["room temperature"]}
Then, plot the distribution of the protein standards on the different sucrose gradient fractions. #####%%%%% output "the different sucrose gradient fractions" are not used
{"action": "plot", "output": "the different sucrose gradient fractions", "reagent": ["the protein standards"], "time": [""]}