User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/25: Difference between revisions
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== | ==Procedure== | ||
#Prepare a 300uM solution of sodium dithionite in 100mL of Tris buffer at pH 8.3 | |||
#Degas solution for an hour | |||
##Nitrogen was bubbled through the solution | |||
#A disposable column was obtained | |||
##GE PD-10 pre-packed column | |||
#The column was cleaned by running 25mL of dionized water through it | |||
#The column was equilibrated by running 25mL of the degassed solution through it | |||
#Once equilibrated, we started running the protein in the following steps | |||
##2.5mL of protein are added to the column (do not collect elute) | |||
##3.5mL of degassed Tris with DTT are added to the column (collect elute) | |||
##25mL of degassed Tris with DTT are added to the column to re-equilibrate (do not collect elute) | |||
##Repeat the steps until all the protein has been run through the column | |||
#After the first collection, a solution of Ruthenium was added to the elute and the following collations (of protein) were added to the same tube | |||
##The ruthenium solution was made up to contain 10X more Ruthenium than protein | |||
#Once all the protein were collected from the column, the tube was covered in foil and placed on the shaker for 4 hours | |||
#During the wait period, the desalting column on the FPLC was cleaned and equilibrated with Tris buffer pH 8.3 | |||
#After 4 hours of reactions, the protein solution was run through the desalting column | |||
#The protein and ruthenium excess were separated using the column and stored in separate tubes | |||
#The tubes were placed in the freezer until further purification next week | |||
Revision as of 11:26, 25 July 2014
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Procedure
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