Polysome profile on sucrose gradient

Alain Jacquier's lab, Institut Pasteur

Materials

50ml tubes (pre-cool)

15ml tubes (pre-cool)

Breaking Buffer

Cycloheximide 50mg/ml (1000X) BE CAREFUL HIGLY TOXIC. SIGMA C-7698 from steptomyces griseus conserved at 4° C. Must be freshly prepared, solve Cycloheximide in ethanol. We need 200 µl per culture (200ml of culture) + 50 µl per 50ml of breaking buffer

Experiment

Sucrose gradient preparation

(For 12 tubes, sucrose gradient from 50% to 10%)

Materials

Complete to 100 ml with H2O

Prepare Buffer 1X

Complete to 100 ml with H2O

Thaw the tubes at RT. Put the tubes on the bench one hour before the centrifugation

Centrifugation and fraction collection

Put about 10 OD260 of cellular extract on the top of the gradient tube (thaw one hour before centrifugation).

Centrifuge the tubes at 4°C for 2h 45min at 39000 rpm in a SW41.

Collect 24 fractions. After 1 min. collect the fractions (about 400µl) corresponding to 30".

Store the tubes at -20°C

Proteins precipitation

Add 50 µl of 100% TCA (in water) to each fraction

Vortex and incubate on ice for 10 min.

Centrifuge at 4°C for 15 min.

Discard the supernatant. Try to eliminate the TCA solution as well as possible.

Resuspend the pellets with 20 µl of sample buffer 1X containing Bromophenol containing 0.2M Tris-base. Sample buffer 1X is prepared from Sample buffer 2X (aliquot at -20°C) and 2M Tris-base. The colour of the solution must be blue.

If the blue solution turns yellow add 1 µl of 2M Tris-base.

Keep the tubes at -20°C.

Load 5 µl on SDS-Polyacrylamide gel