Sonication Buffer (for 1L): Keep Refrigerated
Standard Buffer plus 0.2%w/v sodium cholate added.
1L Standard Buffer
2g Sodium Cholate
for each sample use 200mL sonication buffer and add:
10mg Lysozyme
14uL Beta-mercaptoethanol
.75uL Benzonase

Standard Buffer Low Salt (10L):
5% Glycerol
25mM Hepes
100mM NaCl
0.025% NaAzide
adjust to pH 7.25

Protocol

1)Make sonication buffer and pour 200mL into a Rosette vial. Place Rosette vial into a 2L beaker with ice slurry.
2)Resuspend pellet from 1L culture in the sonication buffer.
3)Sonicate for 30 minutes total (15seconds on; 15seconds off = 15min sonication time) at level 10.
4)Pour lysate into a 250mL centrifuge tube and centrifuge for 20 minutes at 14,500 RPM (~10,000xg). Do next step while spinning.
5)Pour 250mL Standard Buffer into a 500mL centrifuge tube. Add 10mL Nickel resin slurry to tube. Let resin settle to bottom.
6)Remove 250mL tubes from centrifuge and take 100uL to run as "clear" sample on gel.
7)Check that resin has settled well. Pour standard buffer into sink and pour cleared lysate on top of resin bed. Place on roller in cold room and let sample bind to resin for 45min (4oC).
8)Stand tubes up in cold room and let resin settle to the bottom (5-10min or until resin looks settled). Aspirate liquid without disturbing resin bed. Take a "no stick" sample for SDS-PAGE.
9)Pour 250mL 10mM Imidazole in Standard Buffer onto resin bed. Place on roller in cold room and let sample rinse for 30min.
10)Repeat step 8.
11)Repeat step 9.
12)Repeat step 8.
13)Pour 250mL 20mM Imidazole onto resin bed. Place on roller in cold room and let sample rinse for 30min. Get disposable columns/plugs ready.
14)Repeat step 8, but keep pipette and add remaining liquid/resin to the disposable column.
15)Let liquid drip through to waste until liquid is just above the resin bed.
16)Add 10mL 20mM Imidazole to the 500mL tube and rinse the extra resin off walls of tube. Add to column.
17)Repeat step 15.
18)Add 5mL 20mM Imidazole to column.
19)Repeat step 15 and 18 until you have done 5 X 5mL washes with 20mM Imidazole. Make sure you have 50mL conicals labeled and ready.
20)Repeat step 15.
21)Add (#mL resin plus 1mL) 250mM Imidazole to the column. Let drip through until about 1mL remains above the top of resin bed. Plus the column and let it sit for 30min. Prepare dialysis items (cut snakeskin, get clips, add buffer and stir bar to 4L beaker)
22)Unplug column and add remaining volume of 250mM imidazole. (generally we shoot for around 15-20mL total eluate).
23)Add eluate to dialysis membrane and dialyze against Standard Buffer overnight at 4°C.
24)Run gel of samples.
25)Use Amicon Ultra (Millipore) to concentrate if desired.