Percoll Gradient

Percoll Gradient

Contributor: Suprya Jayadev
Date: August 27, 1991

Forming gradient

1) Add 22 ml of light Percoll to a centrifuge tube.

2) Using a 20 G 3 1/2 in spinal needle on a 10 cc syringe, carefully underlay with 6 ml of dense Percoll.

3) Centrifuge tubes at 48,000xg (~20,000 rpm in JA-20 rotor in Beckman J2-21 high speed centrifuge) for 15 mins, 40C.

Fractionation

4) Carefully layer supernatant on top of the preformed gradient; without disrupting the gradient!!

--> In addition to gradient containing sample, should also run a marker gradient loaded with colored marker beads of known density (Pharmacia).

5) Centrifuge samples at 48,000xg , 40C, 15 mins, as before.

6) Collect 1 or 2 ml fractions at 40C by aspiration from the bottom of the gradients through a pipet tip attached to polyethylene tubing and peristaltic pump.

Removing Percoll

7) Should have collected 19 or 38 fractions into 5/8 X 3 in,10.4 ml capacity centrifuge bottles (Beckman # 355603) during the previous step.

--> Determine the distances of bead migration in the marker gradient.

8) Weight balance tubes using Percoll balancing solution.

9) Centrifuge fractions at 180,000xg (~60,000 rpm using the 70.1 Ti rotor in the Beckman L7-65 ultracentrifuge) for 30 minutes.

--> or at 100,000xg for 90 minutes

10) Collect supernatants and discard pellets.

Reagents:

Relaxation buffer (without EGTA):
100 mM KCl
3 mM NaCl
1 mM ATP(Na)2
3. 5 mM MgCl2
10 mM PIPES pH 7.3

Light Percoll (final density 1.040 g/ml)
10 ml 10X relaxation buffer with EGTA
26.4 ml undiluted Percoll (density 1.130)
63.6 ml dH2O
--> relaxation buffer makes Percoll isotonic
--> relaxation buffer should contain 12.5 mmol/l EGTA

Dense Percoll (final density 1.120 g/ml)
3 ml 10X relaxation buffer with EGTA
26.4 ml undiluted Percoll
0.60 ml dH2O

Percoll balancing solution (final density 1.122 g/ml)
5 ml 10X relaxation buffer with EGTA
45 ml undiluted Percoll