Materials

50ml tubes (pre-cool)

15ml tubes (pre-cool)

Breaking Buffer

For 50 ml of breaking buffer:

10 mM Tris-HCl pH 7.4 0.5 ml of Tris-HCl pH 7.4 1M

100 mM NaCl 1 ml of NaCl 5M

30 mM MgCl₂ 1.5 ml MgCl₂ 1M

50µg/ml Cycloheximide 50 µl of cycloheximide 1000X

1 tablet of protease inhibitor (ref ROCHE Complete 20 tablets 1 697 498) store at 4°C

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 200µl per culture (200ml of culture) + 50 µl per 50ml of breaking buffer

Experiment

- Put the tubes for the sucrose gradient on the bench in the evening in order to perform the centrifugation tomorrow morning

- One day before the polysomes extraction, inoculate 200 ml of medium,

- Growth o.n. at 30°C.

- The next morning, measure the OD₆₀₀. It should be at around 0.5. Not higher than 0.6

- Add cycloheximide 1000X to the culture and put the flask on ice.

- Collect the cells in 50ml tube (precooled) by centrifugation for 5 min. at 3500rpm into Jouan at 4°C.

- Resuspend the pellet into 10 ml of pre-cooled breaking buffer.

- Centrifuge the cells in 15ml tube by centrifugation for 5 min. at 3500rpm into Jouan at 4°C.

- Resuspend the pellet into 200 µl of pre-cooled breaking buffer.

- Transfer the cells (final volume of about 600 µl) into an ependorff tube containing about 500 µl of glass beads.

- Vortex for 30“, put the tube for 30″ on ice;

- Repeat 12 times. Put the timer on 12 min.

- Centrifuge at 14000 rpm for 10 min. to eliminate the glass beads.

- Transfer the cells in another ependorff tube and centrifuge again at 14000rpm for 10 min. to clarify the supernatant.

- Measure the OD at 260nm of a 1/500 dilution. It must be around 0.2 at 260.

- Aliquot by 10 OD260 containing fractions if possible and store at –80°C.

- (Usually, half of the polysomal extract from 200 ml of culture is load on the sucrose gradient tubes).

BE CAREFUL: The sample could not be frozen again after thawing.

Sucrose gradient preparation

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

Materials

Beckman tubes Ultra-clear ref 344059 (14X89mm)

50% sucrose

50 g of sucrose solve into:

5 ml of Tris HCl pH 7.4 1M

1.2 ml of MgCl₂ 1M

1.25 ml of NH₄Cl 4M

100 µl of DTT 1M

1 tablet of protease inhibitor

Complete to 100 ml with H₂O

Prepare Buffer 1X

5 ml of Tris HCl pH 7.4 1M

1.2 ml of MgCl₂ 1M

1.25 ml of NH₄Cl 4M

100 µl of DTT 1M

Complete to 100 ml with H₂O

40% sucrose:

25 ml of Sucrose 50% + 6.25 buffer 1X

30% sucrose:

25 ml of Sucrose 50% + 16.65 buffer 1X

20% sucrose:

15 ml of Sucrose 50% + 22.5 buffer 1X

10% sucrose:

10 ml of Sucrose 50% + 40 buffer 1X

Add 2.3 ml of sucrose 50% into Beckman tubes.

Put the tubes at –80°C for 30 min.

Add 2.3 ml of sucrose 40% into Beckman tubes.

Put the tubes at –80°C for 30 min.

Add 2.3 ml of sucrose 30% into Beckman tubes.

Put the tubes at –80°C for 30 min.

Add 2.3 ml of sucrose 20% into Beckman tubes.

Put the tubes at –80°C for 30 min.

Add 2.3 ml of sucrose 10% into Beckman tubes.

Put the tubes at –80°C for 30 min.

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

Centrifugation and fraction collect

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

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

Collect the fraction:

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)

Vortex and incubate on ice for 10 min.

Centrifuge at 4°C for 15 min.

Discard the supernatant. Try to eliminate the TCA solution.

Resuspend the pellets with 10 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