Develop cells for at least 3 hrs to minimize protease activity. Cell
are resuspended in DB to 2-4 x 107/ml and shaken at 120 rpm at 22o
for 3-6 hrs.
Cells were harvested and washed with TEB (40 mM Tris-HCl, pH 7.5, 50 mM
NaCl, 2 mM EDTA). They were finally resuspended in TEBP (TEB plus various protease
inhibitors: 1x PIC I and 1x PIC II) to 2 x 108 /ml.
CHAPS powder was added at 20 mg/ml. After gentle mixing, the lysed
cells were kept on ice for 3-5 minutes and then sucrose crystals were added to 55% final
concentration (calculate sucrose grams: 2 x cell volume (mls) / 2.7).
They were loaded under a 20-45% step sucrose density gradient and
centrifuged at 150,000g for 12 hrs. For SW 41 rotors, use 3 ml 20%, 4 ml 45 % and 5 ml
sample volumes. The CHIFF band was collected by capillary, diluted 4 fold with TEB and
spun down. The CHIFF pellet is finally resuspended to 5 x 108 cell
equivalent/ml in TBP (TEBP without the EDTA) with 30% sucrose and then stored frozen at
Sucrose crystals (Sigma) were added to the CHAPS lysate to a final concentration of 55%
(w/v). A 8 ml linear 20-45% sucrose gradient (in TEBP) was formed over 4 mls of this
sucrose-containing CHAPS lysate.
The gradient was centrifuged at 36,000 rpm for 14-16 hours in a Beckman SW 41 rotor at
8oC. A thin, white band in the middle section of the gradient was collected,
diluted 5 fold with TEB, and centrifuged in a SS34 rotor at 12,000 rpm for 15 minutes.
This pellet preparation was designated CHIFF (CHAPS
insoluble floating fraction). In some cases, the gradient was pumped from the bottom into 12-15
fractions (~0.8 ml/fraction) and the fractions analyzed by immunoblot. In later
experiments, we found out that by replacing the linear gradient with a step gradient of 4
ml 20% and 4 ml 45% sucrose, we could obtain essentially the same CHIFF preparation.
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