Day 2
1. Add 1ml of 1000x carb to 1L LB then inoculate with all of overnight culture.
2. Grow in 37 degree shaker until log phase (~3 hours depending on mutant).
3. Check cells for log phase growth by using spectrophotometer until OD(600) ~ 0.5-1.0. (grow till OD(600) ~0.2 using the small tabletop spectronic spec)
4. Add 1ml 1000x IPTG to induce expression.
5. Grow in 37 degree shaker for 4-6 hours.
6. Spin down cells using 1L centrifuge (4000 rpm for 20 minutes).
7. Remove supernatant. At this point you may place cells in -20 degree freezer or go on.

Day 3
1. Add 30mls 1x binding buffer to cells.
2. Add 300ul of 100x PMSF and 100x benzamidine (protease inhibitors).

100x PMSF = 17.4 mg/ml in isopropanol
100x Benzamidine = 39.2 mg/ml in H2O or isopropanol.

3. Add spatula tip of Dnase (~250ug) and a very small spatula tip of Rnase (~50ug).
4. Resuspend cells and quick freeze using Dry ice and EtOH to lyse cells. Thaw solution and repeat quick freeze 2x.
5. Incubate at room temp for 15-30 minutes until all Dna gets digested (viscosity of solution will decrease to viscosity of water).
6. Spin down cell lysate in GSA or SS34 tubes (10000 rmp for 20 minutes).
7. Separate supernatant into a labelled falcon tube.
8. Run 10 ul supernatant and pellet (very small pipet tip smear into 10 ul water) on a 14% Tris-tricine gel to check if protein expressed into supernatant or into inclusion bodies (pellet).
9. You may freeze pellet and supernatant to purify protein later.

Purification
Day 1

1. Check gel to see where protein was expressed

If expressed in pellet use 3M GuHCl (practical grade, filtered, purified) in all buffers used for purification (denatured conditions). Resuspend the pellet in 0.5% TritonX-100 then spin @12,000 rpm for 20 minutes (this cleans up the inclusion bodies). Remove the supernatant and solubilize the pellet with 6M GuHCl (10mls) in 1x binding buffer. Use a spatula to break up the pellet and rock at room temp until most is solubilized. Spin down solubilized pellet in SS34 (12000rpm for 30 minutes) to remove unsolubilized junk. Save supernatant to load onto column.

2. wash column (20ml bed volume) with 2 bed volumes of water.
3. charge column with 1x charge buffer (2 bed volumes)
4. wash column with 2 bed volumes binding buffer (if protein was in the pellet don't forget to use denaturing conditions (3M GuHCl in all the buffers).
5. load supernatant (if protein was in pellet load the solubilized pellet onto the column - the column should have 3M GuHCl 1x binding buffer in it.)
6. Save flowthrough in case protein does not bind to column.
7. Wash column with 10 bed volumes of 1x binding buffer with 0.5% Triton X100, save flowthrough.
8. Wash column with 10 bed volumes of 1x wash buffer, save flowthrough.
9. Elute protein with 1x strip buffer. Use fraction collector to collect fractions with protein. (all protein usually elutes in ~50mls strip buffer).
10. Collect fractions with protein.

Day 2
Desalt protein for biophysical characterization
Prepare protein to load on G25 desalt column. (must concentrate to < 10mls)
1. Concentrate protein to < 10 mls using Amicon concentrator (YM3 membrane 43mm, using Nitrogen tank in cold room) - this takes a long time ~3 hours. Make sure you keep a good eye on the concentrator so it does not dry out. If it does dry out, solubilize the protein by adding 6M GuHCl ( < 10 mls) and stir for 10 minutes.
2. Equilibrate desalt column with 2 bed volumes of 50mM NaPi pH7 (1 bed volume = 200mls). If your protein is in denaturing conditions (3-8M GuHCl) use column equilibrated with 3M GuHCl, 50mM NaPi pH7.
3. Load sample, then run 50mM NaPi pH7 through column (if protein is in denaturing conditions run 3M GuHCl, 50mM NaPi pH7 through column). Sample should come out of column after ~60 mls of flowthrough. Use fraction collector to collect fractions with protein.
4. * Optional-Concentrate protein with Amicon concentrator (to ~15 mls). Depending on expression levels, you should have ~15 mls of ~300-900uM protein. Check concentration of protein using Aviv spectrophotometer (absorbance at 280nm).