User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/28: Difference between revisions
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== | ==Clean and store Desalt column== | ||
#Run 150mL water through the column | |||
#Run 150mL 20% Ethanol solution throughout the column | |||
==Make buffers for FPLC== | |||
#Prepare 400mL 20% Ethanol | |||
##80mL 200 poof ethanol in 320mL FPLC water | |||
#Filter 1L H20 and prep for autoclave | |||
#Prepare 1L 20mM Tris pH 8.3 | |||
##Filter and prep for autoclave | |||
#Place buffer and water in autoclave at 121C for 1 hour | |||
==Prepare Protein solution== | |||
#Thaw tube on lab bench | |||
#Place solution in centrifugal device with 30mL pH8.3 20mM Tris buffer | |||
#Centrifuge for 30 minutes at 3000g | |||
#Repeat above steps but this time for 1 hour | |||
#Centrifuge until final volume is 10mL | |||
==Equilibrate Q-Sepharose column== | |||
#Run 25mL H2O through the column | |||
#Run 25mL 20mM Tris pH 8.3 | |||
#Column is ready for protein | |||
==Purification fo Protein== | |||
#Inject protein into column | |||
##Collect elute during injection | |||
#Run a gradient 0-300mM NaCl in Tris over 10 minutes | |||
##Collect fractions | |||
#Combine desired fractions into single tube | |||
#Clean column with 20mL 1M NaCl | |||
#Re-equilibrate column with 20mM Tris pH 8.3 | |||
#Concentrate and desalt fractions using centrifugal device | |||
##Add 20mM Tris to fractions twice to remove salt | |||
##centrifuge until final volume is 10mL | |||
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Revision as of 08:53, 28 July 2014
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Clean and store Desalt column
Make buffers for FPLC
Prepare Protein solution
Equilibrate Q-Sepharose column
Purification fo Protein
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