NWASp protein purification using UREA (October 4, 01)
1) Get 1uL or less of plasmid pBH8 (NWASp mino
acids 392-501). Transform electrocompetant BL21 or chemically competant-
I usually transform a few platefuls since we innoculate
18liters worth of cells.
2) Grow in TPM until OD600 is between 0.4 and
0.6 (the doubling time of most E. coli strains is 20 minutes). Use this aliquot to take the OD to make a gel
sample of pre-induction of the cells. Add IPTG to 0.25mM from a 1M stock and
grow 2 more hours at 37°C with shaking.
3) Take OD600 of cells after induction and use
this for a gel sample post-induction.
4) Harvest the cells by centrifugation in the
RC3B at 3000rpm for 15 minutes, Use the 1 liter bottles. Decant the supe and
keep combining the pellets until all in a few tubes, preferably one tube.
5) Resuspend the cells in 10mM Tris, 0.5mM EDTA, pH 7.0
(200mls) and spin in 50mL falcon tubes.
Decant supe, snap freeze the cells in
Nitrogen.
6) Weigh the cell pellet. The buffers with
UREA must be pHed right before use since Urea
dissociation can interfere with pH. Thaw
cell pellet in Buffer B (1Liter Buffer B= 100mM NaH2PO4, 10mM TrisHCl, 8M Urea, 500mM KCl, pH
8.0 with NaOH.) Use 5mL buffer per gram of cell weight.
7) Place on shaker O/N at room
temperature. Spin at 10,000rpm for 20
minutes in Sorvall centrifuge.
8) Get Nickel resin ready. Use about 20-25mLs
of resin. You may need to recycle some
if it is not available fresh.
9) Batch bind to Nickel resin (NiNTA) for 2 hours at room temperature. Place into a
column, collect flow through and make a gel sample of this. Save flow through so that in case all of it
did not bind you have more.
10) Wash with buffer B (use 4 column volumes so
about 100mLs). Wash in 4 column volumes of
Buffer C (100mM NaH2PO4, 10mM Tris HCL, 8M urea, pH 6.3 using HCL.
11) Wash with 3 column volumes of 1X Binding Buffer (For 200mLs of 8X Binding buffer: 40mM Imidazole, 4M NaCl,160mM Tris, pH 7.9. Take a gel sample of last few microliters of wash.
2M NaCl 80mM Tris-HCL, pH 7.9 Water to 100mLs). Collect 10mL elutions in glass tubes or falcon tubes, make gel samples and run a gel.