Team:Washington/Notebook/2mL purification

==== Supernatant Protein Purification, 2mL ====
#Inoculate 50mL culture of TB with ~750uL overnight culture
#Grow a 37c until OD600: 0.4
#Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
#Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
#Prep NiNTA columns 
##Place micro-centrifuge columns in collection tubes
##In micro-centrifuge columns add 200uL NiNTA beads
##Add 500uL PBS, aspirate to thoroughly rinse columns 
##Spin columns with collection tubes for 30sec at 500rpm
#Transfer 2mL of growing culture to eppendorf tube
##Spin tube for 20min at 8000 rpm
##Remove supernatant, carefully as to not disrupt the pellet and set aside
#Bind Protein to column
##Add 500uL supernatant to the column
##Spin for 30sec at 500rpm
##Discard flow through
##Repeat 1-3 until all supernatant has run though the column
#Wash column
##Apply 500uL PBS to column, aspirate to suspend beads
##Spin column for 30sec at 500rpm, discard flow through
##Repeat 1-2
#Elute protein off of column
##Make PBS with 1mg/mL BSA and 100uM imidazole
##Add 200uL PBS + 1mg/mL BSA + 100uM imidazole to column aspirate to mix beads
##Let sit for 2 min
#Place Column in clean collection tube
#Spin column for 5min at 500rpm 
#FLOW THROUGH IS YOUR PURIFIED PROTEIN