Knight:Purification of His-tagged proteins/Denaturing: Difference between revisions

From OpenWetWare
Jump to navigationJump to search

Knight:Purification of His-tagged proteins/Denaturing (view source)

Revision as of 12:00, 16 July 2006

1,062 bytes added ,  16 July 2006
→‎Procedure
Line 42: Line 42:
#*''I stored the pellet at -80°C.''
#*''I stored the pellet at -80°C.''
#Thaw for 30 mins.
#Thaw for 30 mins.
#*''I thawed at room temperature since the next step happens at room temperature.''
#*''Thaw at room temperature since the next step happens at room temperature.''
#*''The Qiagen protocol calls for 15 mins, but it was still frozen after 15 mins so I let it thaw for 30 mins.''
#*''The Qiagen protocol calls for 15 mins, but it was still frozen after 15 mins so I let it thaw for 30 mins.''
#Transferred to 2mL eppendorf tube.
#Transferred to 2mL eppendorf tube.
#Resuspend in 1mL Lysis Buffer (see above).
#Resuspend in 1mL Lysis Buffer (see above).
#Incubate cells with agitation for 1 hr at room temperature.
#Incubate cells with agitation for 1 hr at room temperature.
#*''Used an orbis shaker on the bench to do this temp (usually kept in 37° incubator).  Note that the shaker moves during shaking.''
#*''Use an orbis shaker on the bench to do this temp (usually kept in 37° incubator).  Note that the shaker moves during shaking.''
#Centrifuge lysate at 10000 x ''g'' for 30 mins at room temperature.
#Add 600 μL lysis buffer to Ni-NTA column to equilibrate.
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid to remove equilibration buffer.
#Save 20 μL cleared lysate.
#Load 600 μL cleared lysate to Ni-NTA column.
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
#*''Repeat this step to load the rest of my cleared lysate?''
#*''Save flow through.''
#Add 600 μL wash buffer to Ni-NTA column.
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
#*''Save flow through.''
#Add 600 μL wash buffer to Ni-NTA column.
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
#*''Save flow through.''
#Tranfer to clean 1.5mL eppendorf tube.
#Add 200μL elution buffer.
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
#*''Most of the protein should elute in this elution step.''
#Tranfer to clean 1.5mL eppendorf tube.
#Add 200μL elution buffer.
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
#*''Just in case.''


==Notes==
==Notes==
Reshma P. Shetty
7,287

edits

Retrieved from "https://openwetware.org/wiki/Special:MobileDiff/50720"

Navigation menu