• Prepare 12L of LB and preheat at 37oC overnight
• Grow 100ml culture of LB+CAM+(selective marker) overnight at 37oC @200rpm
• Next day add respective antibiotic (but NOT CAM) to the 12L's and 25ml of culture per 2L of LB and shake at 37oC @200rpm until an O.D. @590nm of 0.6 is reached.
• Once the O.D is reached, save 1ml of culture in an eppendorf tube, spin it at max for 1min, remove the supernatant and resuspend in 33ul of 2x Loading buffer, label it "pre-induction" and store at -20.
• Prepare buckets of ice water
• Place the 12L LB cultures in the water and add 1ml of 1M IPTG per 2L of LB and shake for 3hrs at room temp @ 150rpm
• Take a 1ml sample of culture, spin at max for 1minute, remove supernatant and resuspend in 100ul 2X Loading buffer, label as post induction..
• Pour the cultures into 1L plastic bottles and spin in Sorvall @4000rpm for 30min.
• Keep on ice!!! Discard supernatant and resuspend in 10ml  of prechilled buffer A(+Leupeptin+ Pepstatin+Aprotenin+PMSF+10mM beta-ME --> add fresh, and keep this solution separate from the stock of buffer A)
• Pool the suspension into a few Falcon tubes and imerse into liquid N2, and store at -80 until the next day.

Day2:

• Retrieve tubes from -80 and thaw in a beaker filled with water.
• Prepare the flex column, and wash with buffer A + 10mM Imidazole ~50-100ml.
• Precool glass beakers, flasks, 50ml plastic culture tubes and the SS-34 Rotor.
• Place the cell lysates into precooled beakers, place them on ice and sonicate using the large sonication head (keep sonicator on high, and continuous voltage).  Sonicate cells 4 times, 1min the first time and 30sec the other times.  KEEP THE CELLS ON ICE EVEN DURING SONICATION!!  Make sure there are no cell clumps left and that the lysate is no longer viscous (due to the shearing of the genomic DNA).
• place the lysates into the precooled 50ml tubes and spin using the Sorval SS-34 rotor @17K for 20minutes.
• Discard the pellets and place the supernatant(s) into new precooled tubes and spin using the SS-34 rotor for 10min @ 17K.
• Discard the pellets and pool supernatant(s), pour into precooled bottles.
• Back-load the lysates onto the flex columns at a rate of 0.5-1ml/min
• Wash further with 100 ml buffer A + 10mM imidazole + beta-ME
• Wash with 50 ml buffer B (to rid the phosphates from the sample)
• Take the column out of the cold room, remove carefully the flow adapter, allow all the excess buffer to come out.
• Take a 500ul sample of the column beads itself and label as beads, store at -20.
• Then add and collect 1ml of buffer B + 100mM imidazole (protein eluate)
• Using a microtiter plate, assay for protein levels using 10ul of protein sample from each fraction + 100ul of bradford solution.
• Run 5ul of each fraction on SDS gel..
• Keep the protein fractions on ice and store in the cold room overnight
• Stain the gel overnight with coomasie blue.
• Pool positive fractions and freeze in liquid nitrogen, store at -80oC

• Buffer A: PBS +100mM NaCl + 1% Triton X + 10 mM imidazole
• Buffer B:  50mM Tris + 100mM NaCl