Preparation of Eleven Step Sucrose Gradients (54%-18%) for Cell Fractionation
Materials
Buffer slution: 10 mM Hepes pH7.4, 2 mM MgCl2 and 10mM NaN3
High grade sucrose
5ml Gilson pippette and tips (P-5000)
250 or 500 ml bottles
50 ml Falcon tubes
polyproplyne centrifuge tubes
Ice
Procedure
Prepare a stock of 65% sucrose in buffer solution: Dissolve 162.5g sucrose in 250ml buffer solution.
Make 25ml solution each in 50ml tube for the steps in the gradient according the following table:
Sucrose percentage in steps
Volume of sucrose stock (ml)
Volume of Hepes/MgCl2 buffer (ml)
65
use stock dirrectly
0
54
20.77
4.25
50
19.23
5.77
46
17.69
7.31
42
16.15
8.85
38
14.62
10.38
34
13.08
11.92
30
11.54
13.46
26
10.00
15.00
22
8.46
16.54
18
6.92
18.08
mix the step solutions and chill on ice prior to use.
Pack a polyproplyne tube on ice. To it, using P-5000, add 1.7ml of 65% sucrose. Then on top of this sequentially layer 3.4 ml of 54%, 50%, 46%, 42%, 38%, 34%, 30%, 26%, 22% and 18% sucrose. Make sure that you add the sucrose to the center of the tube and do so gently. You don't want to get a lot of mixing.
When the gradients are made they can be stored on ice until they are needed.