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
2M KOAc
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