Plant polysome purification

Polysome purification and fractionation

(Based on Davies & Abe, 1995, Methods in Cell Biology, Vol 50, Chapter 15)

1. Prepare 4.6ml 15-60% sucrose gradients in 5ml polyallomer tubes (Beckman #326819) by pipetting 2.3ml of 60% sucrose in buffer B followed by 2.3ml of 15% sucrose in buffer B (slowly, drop by drop, against the wall). Cap the tubes with rubber stoppers and lay tubes horizontally for 3h @RT for sucrose to diffuse (or for 2h if making 10-40% gradient). Place the tubes again vertically and allow to cool in refrigerator for at least 30 minutes.  Keep the gradients (vertically, with stoppers on) @4C until needed for up to 5-6 days.

2. Grind 0.6-1g of tissue in liquid nitrogen using a mortar and a pestle. Transfer the powder to a second ice-cold mortar filled with 6.5ml of ice-cold buffer U and continue grinding.

3. Filter the extract through a Miracloth into a small (50ml) beaker to remove cell debris, squeeze out the fabric to recover the total of ~5ml extract. Divide the extract into 4-5 pre-chilled microfuge tubes.

4. Centrifuge the samples in a cold microfuge at top speed for 10 minutes to collect the remainder of cell debris.

5. Transfer and combine the supernatants in a pre-chilled 15-ml tube (plastic blue cap tubes are OK) to check the volume of the extract recovered, being careful not to transfer any of the pellet.  If volume is lower than 5ml, add more ice-cold buffer U.

6a. Direct Layering. Layer 3 x 200ul of the cleared extract onto three identical 15-60% sucrose gradients made in buffer B (see step 1).  Do it slowly, drop by drop, against the wall.  Spin the samples in an ultracentrifuge in a SW50.1 rotor (or equivalent) at 45K for 1h 15min @4C.  Read the polysomal profiles on an ISCO machine at 254nm.  Collect the fractions if desired (for northern, western, etc.).  Collecting by the number of drops rather than by volume may be more practical (~12 fractions of 8 drops each, with the total volume of each fraction increasing as sucrose concentration rises).

6b. Polysome pelleting. Layer 4.3ml of the cleared lysate onto a 0.5ml 50% sucrose pad (prepared in buffer A) placed on the bottom of a 5ml polyallomer tube. Spin the sample in an ultracentrifuge in a SW50.1 rotor (or equivalent) at 45K for 3h @4C. Invert the tubes to discard the supernatant (the semi-transparent polysomal pellet is on the very bottom of the tube and may be hard to see) and sit them up-side-down on a paper towel for ~30 seconds to drain.  Rinse the walls of the tube with 1ml of ice-cold water, and invert the tube to dry on a paper towel for 30 seconds.  Freeze the polysomal pellets at -80C until needed or directly proceed to the next step.

7. The polysomal pellets can now be resuspended in an appropriate volume of water (for RNA extraction), buffer U (for secondary polysomal profiles), or other buffer of choice dependent on the intended downstream application.  To aid pellet resuspension, insert a pre-chilled glass or blue plastic rod into the ultracentrifuge tube and vortex the sample vigorously in the buffer of choice.  Example: Resuspend the pellets in 450ul of ice-cold water (insert a glass/plastic rod, vortex 3-5 times, place on ice). Split the sample into two.  To one half (~200ul), add 1 volume of 2 x buffer U (1x is OK, too), vortrex, and layer 2 x 200ul onto two identical 15-60% sucrose gradients (see step 1; then proceed as described in step 6a).  To the second half of the sample (200ul), add 700ul of buffer RLT, split into two, and proceed with the RNeasy cleanup protocol (Qiagene).

Buffers and recipes

Stock solutions:

80% RNase-free sucrose in water

1M Tris-HCl, PH 7.5

2M Tris-HCl, pH 8.5


1M MgOAc

O.5M EGTA, pH 8.0

Polyoxyethylene 10-tridecyl ether (PTE, a non-ionic liquid detergent, Sigma P2393; if solidified, warm up in a microwave to >40C to liquidify)

Sodium deoxycholate (DOC, an anionic detergent (powder), Sigma 30970)

Heparin (sodium salt, Sigma H9399)

5 x Buffer A (1M Tris-HCl, pH 8.5, 250mM KOAc, 125mM MgOAc) (200ml):

100ml 2M Tris-HCl, pH8.5

25ml 2M KOAc

25ml 1M MgOAc

50ml water

10 x Buffer B (500mM Tris-HCl, pH 7.5, 250mM KOAc, 100mM MgOAc) (200ml):

100ml 1M Tris-HCl, pH7.5

25ml 2M KOAc

20ml 1M MgOAc

55ml water

1x Buffer U (200mM Tris-HCl, pH 8.5, 50mM KOAc, 25mM MgOAc, 2mM EGTA, 2% PTE, 0.8% DOC, 0.1mg/ml heparin) (250ml):

~200ml pre-warmed water

2g DOC; stir to dissolve

5ml PTE

25ml 1M Tris-HCl, pH 8.5

6.25ml 2M KoAc

6.25ml 1M MgoAc

1ml 0.5 M EGTA

Water up to 250ml

25mg heparin

60% sucrose in Buffer B (200ml):50ml:

20ml 10 x Buffer B            5ml

150ml 80% RNase-free sucrose               37.5ml

30ml water         7.5ml

40% sucrose in Buffer B (200ml):50ml:

20ml 10 x Buffer B        5ml

100ml 80% RNase-free sucrose        25ml

80ml water        20ml

10% sucrose in Buffer B (200ml):50ml:

20ml 10 x Buffer B         5ml

25ml 80% RNase-free sucrose6.25ml

155ml water        38.75ml

15% sucrose in Buffer B (200ml):50ml:

20ml 10 x Buffer B         5ml

37.5ml 80% RNase-free sucrose         9.375ml

142.5ml water         35.625ml

50% sucrose in Buffer A (200ml):50ml:

40ml 5 x Buffer A        10ml

125ml 80% RNase-free sucrose        31.25ml

35ml water        8.75ml

Chase solution for ISCO machine (60% sucrose, 20% KCl) (500ml):

300g sucrose (regular is OK)

100g KCl

Water up to 500ml