SW28 Rotor - 25 hr, 24,000 rpm, 4oC. Load 2-3 ml sample.
or
SW41 Rotor - 17.5 hr, 30,000 rpm, 4oC. Load 0.2 ml sample.
Preparation of gradients:
Biocomp Gradient Maker - put SW28 or SW41 tube in metal device and add 30% glycerol solution to line. Then layer 10% glycerol solution on top of the 30% to the top of the tube. Insert cap sealing device - avoid air bubbles. Run on Biocomp using specific program for the SW28 or SW41 rotor. REmove cap and layer sample.
Tap gradients in ISCO Fractionator:
SW28 - Program A. 3 ml/min.
Collect 3 ml fractions. Delay = 11 sec.
SW41 - Program B, 1.5 ml/min, 30
sec/fraction. Delay = 22 sec.
Gradient buffer:
25 mM HEPES buffer (pH 7.5)
60 mM KCl
10 mM MgCl2
Add glycerol
to final concentration of 10% or 30%.
For 100 ml:
5 ml 0.5 M HEPES (pH 7.4)
2 ml 3 M KCl
1 ml 1 M MgCl2
10 ml or
30 ml glycerol
Add water to 100 ml