Team:Washington/Notebook/IMAC protocol

BioRad Micro Column Protein Prep Protocol

 * Day 0: Clone gene of interest into vector of interest using standard cloning techniques.
 * Day 1: Overnights
 * Pick a single colony from plate and inoculate 1mL fresh media, with proper antibiotic, in 15mL culture tube
 * Place lid on tube as not to form an air tight seal
 * Shake at 37C for 16-24 hours at 200+ rpm
 * Day 2: Expression
 * Inoculate 50mL media with 500uL over night, in 250mL flask
 * Shake 250mL flask at 37C at 200+ rpm
 * Monitor OD600 of culture until OD600 ~0.4
 * Use 200uL of culture and 800uL water in 1mL cuvette
 * Read OD600 and multiply by 5 t get original OD600
 * Should take ~2-3 hours
 * INDUCE culture once culture gets to OD600 ~0.4 with IPTG so that the final concentration of IPTG is 0.5mM (ie 25uL 1M IPG for 50mL culture)
 * Place in shaker 24-36hr at 18C.
 * Day 3: Purify (~3.5 hours)
 * Pellet Cells (0.5hr)
 * Spin down cells at 4000rpm for 20 min in 50mL falcon tubes, discard supernatant
 * Cells can be stored in -20C until ready for purification
 * Lyse (0.5-1hr)
 * Add 1mL of lysis buffer, re-suspend, transfer to a 2mL eppindorf tube
 * Continue to incubate at room temperature for 20min, mixing with plate mixer
 * If worried about protein stability then incubate in cold room for 1hr on rocker
 * Pellet Insoluble Fraction (0.5-1hr)
 * Spin the lysis for 30-60min at 15000rpm (spin longer if supernatant is not clear, but 1hr should be plenty!)
 * Transfer supernatant to a fresh tube (~1800uL)
 * Filter (0.1hr)
 * Run supernatant through a 0.4micron filter to remove any remaning particules that would clog columns.
 * Bind Protein (0.5hr)
 * Add 100uL of NiNTA Agarose 50% slurry to each column (this is the Ni-beads)
 * Add 600uL of diH2O to each column
 * Spin 500rpm for 10s, discard flow through
 * Add 500uL of wash buffer, spin, discard flow through
 * Add 500uL of supernatant, spin, discard flow through
 * Repeat previous step until all supernatant has passed over column (~4x)
 * Wash Protein (0.5hr)
 * Add 500uL of wash buffer, spin, discard flow through
 * Elute Protein (0.2hr)
 * CHANGE TO FRESH COLLECTION TUBE
 * Add 200uL of elution buffer pipette up and down to mix the beads
 * Incubate for 2min the spin at 500rpm for 1min
 * THE FLOW THROUGH IS YOUR PURIFIED PROTEIN!
 * Recover the beads for re-use (~45min)
 * Extract beads using 200uL diH2O and transfer to 15mL falcon tube
 * Store columns in dry place for later use after thoroughly washed out with water. It is fine to just run tap water over the columns after beads have been removed
 * Spin down 800rpm for 5min, then decant supernatant
 * Fill falcon tube with 100mM EDTA, and re-suspend beads
 * Spin down 800rpm for 5min, then decant supernatant
 * Fill falcon tube with diH2O, and re-suspend beads
 * Spin down 800rpm for 5min, then decant supernatant
 * Fill falcon tube with 100mM NiSO4, and re-suspend beads
 * Spin down 800rpm for 5min, then decant supernatant
 * Fill falcon tube with diH2O, and re-suspend beads
 * Spin down 800rpm for 5min, then decant supernatant
 * Add 20% ethanol until a 50% slurry has been reached.
 * Store at 4C


 * Buffer Examples (Alter to fit your protein's ideal buffer)
 * Lysis Buffer
 * 1.5x BugBuster (Solubilizes cell wall)
 * 1mg/ml lysozyme (Helps break cell wall)
 * 0.1mg/ml DNAseI (chews up DNA)
 * 1mM PMSF (Protease Inhibitor)
 * 1x PBS
 * 30mM Imidizole


 * Wash Buffer
 * 1x PBS
 * 30mM Imidizole
 * BUGBUSTER Lysis Buffer
 * 1x PBS
 * 1mM PMSF
 * 2x Bug Buster
 * 2mg/mL lysozyme
 * 0.2mg/ml DNAse
 * Elution Buffer
 * 1x PBS
 * 250mM Imidizole