Team:Washington/Notebook/2mL purification

Supernatant Protein Purification, 2mL

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