Isolation of Ribosomes and Polysomes

Preparation of Cell Extracts

• If harvesting ribosomes from cultured cells, start at Step 1. If using mammalian or plant tissue, start at Step 3. Carry out all steps after cell harvesting on ice or at 4°C. Use sterile and/or RNase-free disposable plasticware where possible.

• 1. Culture desired cell type in appropriate medium. Harvest by centrifugation.

  • Generally, a 4-L culture of E. coli yields ∼7.5 g of cell paste. The cell paste may be frozen in a dry ice/ethanol bath immediately after harvesting and stored at −80°C.

  • If high yields of polysomes are desired, preserve the polysomes by chilling the culture rapidly and adding 100 µg/mL of chloramphenicol (for prokaryotes) or 100 µg/mL of cycloheximide (for eukaryotes) just before harvest.

• 2. Resuspend 5–10 g of fresh or frozen cell paste from Step 1 in the appropriate homogenization buffer (see Step 3) at a 1:2 (w:v) ratio. Add RNase-free DNase to a final concentration of 2 µg/mL and set aside for 20 min at 4°C.

  • Vanadyl ribonucleoside complex can be added to any buffer at a concentration of 10 mm to inhibit RNases. If doing so, omit EDTA from the homogenization buffer.

• 3. Disrupt the cells as follows.

  For Prokaryotic Ribosomes

  • i. Disrupt cells in a prechilled French press at 16,000 pounds per square inch (psi) in French press buffer for prokaryotic 70S ribosomes.

    • Follow the manufacturer's instructions for safe and proper operation of the French press. Generally, once the predetermined pressure reading is reached, while maintaining the psi, open the flow valve to allow the liquid to flow from the sample outlet. Adjust the flow rate to ∼15 drops per minute. Collect the lysate in a flask kept on ice.

  For Prokaryotic Polysomes

  • ii. Disrupt cells in a prechilled French press at 13,800 psi in French press buffer for prokaryotic polysomes.

    • Follow the manufacturer’s instructions for the French press (see Step 3.i).

  For Mammalian Tissue Ribosomes and Polysomes

  • iii. Mince the tissue and resuspend in 2 volumes of mammalian ribosome homogenization buffer. Homogenize the suspension in a glass homogenizer using eight to 10 strokes with a motor-driven loose-fitting Teflon pestle.

    • Determine the amount of starting material empirically and based on availability. This disruption is optimized for tissue such as liver. If another type of tissue or cultured cells will be used, it is important to optimize the homogenization conditions including the buffer; in most cases a detergent lysis may be required.

  For Mammalian Reticulocyte Ribosomes

  • iv. Resuspend cells in 2 volumes of reticulocyte homogenization buffer. Homogenize the suspension with three strokes using a glass homogenizer and a hand-held loose-fitting Teflon pestle.

  For Plant Cytoplasmic Ribosomes and Polysomes

  • v. Wash 25–100 g of plant tissue in RNase-free H₂O at room temperature.

  • vi. Freeze tissue in liquid nitrogen and macerate to a fine powder using a cold mortar and pestle.

  • vii. Resuspend the powder in 2 volumes of cold plant extraction buffer. Homogenize the suspension in a blender by applying 15-sec pulses at 1-min intervals. Do this four times.

  • viii. Pass the homogenate through two layers, and then eight layers, of cold DEPC-treated cheesecloth.

  For Chloroplast Ribosomes

  • ix. Wash 250 g of plant leaves in RNase-free H₂O.

  • x. Suspend the leaves in 250 mL of cold chloroplast homogenization buffer. Homogenize the suspension in a blender for 45 sec.

  • xi. Filter the suspension through a cold DEPC-treated cheesecloth.

  • xii. Clarify the extract by centrifuging at 360g for 5 min at 4°C. Discard the pellet.

  • xiii. Pellet the chloroplasts by centrifugation at 1200g for 15 min at 4°C.

  • xiv. Wash the pellet with chloroplast homogenization buffer and repellet the chloroplasts by centrifugation at 1200g for 15 min at 4°C.

  • xv. Disrupt the membranes and lyse the chloroplasts by resuspending the chloroplast pellet in 50 mL of chloroplast lysis buffer containing either sodium deoxycholate (0.5% final concentration) or Triton X-100 (1.0% final concentration).

• 4. Clear the lysate of cell debris by centrifugation at 4°C using an SS-34 rotor at the following speeds and times.

  For Prokaryotic Ribosomes

  • i. Centrifuge at 30,000g (15,800 rpm) for 30 min. Save the supernatant and proceed to Step 5.

  For Prokaryotic Polysomes

  • ii. Centrifuge at 7700g (8000 rpm) for 8 min. Save the supernatant and proceed to Protocol: Purification of Polysomes (Rivera et al. 2014b).

  For Mammalian Tissue Ribosomes and Polysomes

  • iii. Centrifuge at 20,000g (12,900 rpm) for 10 min. Save the supernatant and proceed to Step 5.

  For Mammalian Reticulocyte Ribosomes

  • iv. Centrifuge at 20,000g (12,900 rpm) for 10 min. Save the supernatant and proceed to Step 5.

  For Plant Cytoplasmic Ribosomes and Polysomes

  • v. Centrifuge at 1076g (3000 rpm) for 7 min.

  • vi. Add 0.1 volume of 20% Triton X-100 to the supernatant and centrifuge at 17,210g (12,000 rpm) for 20 min. Save the supernatant and proceed to Step 5.

  For Chloroplast Ribosomes

  • vii. Centrifuge at 26,000g (14,700 rpm) for 20 min. Save the supernatant and proceed to Step 5.

Isolation of Ribosomes and Polysomes

• 5. Gently layer the crude lysate from Step 4 at a 1:1 (v:v) ratio over the appropriate sucrose cushion (see below) using a pipette. Take care to do this slowly so that a sharp interface is obtained between the cushion and the less-dense layer above. Then centrifuge at 4°C in the Beckman fixed-angle Type 50.2 Ti rotor per the guidelines below.

  For Prokaryotic Ribosomes

  • i. Pellet the 70S ribosomes by centrifugation at 100,000g (28,800 rpm) for 16 h over the high-salt sucrose cushion buffer for prokaryotic ribosomes.

    • This step involves long centrifugation times to bring down 70S ribosomes as well as dissociated 30S and 50S ribosomal subunits. Minimal centrifugation conditions can be used to primarily pellet 70S rather than 50S or 30S subunits.

  • ii. If necessary, repeat two or three times until a clean pellet is obtained.

    • For methanogens, halobacteria, and eocytes (sulfur-metabolizing hyperthermophiles), at least two high-salt washes are necessary to adequately remove the nonribosomal debris.

  • iii. Use, store (at −80°C), or further purify the ribosome-containing pellet as desired.

    • For further purification of the 70S ribosomes, proceed to Protocol: Purification of 70S Ribosomes (Rivera et al. 2014a).

  For Mammalian Ribosomes and Polysomes

  • iv. Centrifuge at 100,000g (28,800 rpm) for 24 h over the mammalian sucrose cushion buffer.

  • v. Use the pellet containing polysomes and monosomes or store at −80°C.

  For Plant Cytoplasmic Ribosomes and Polysomes

  • vi. Centrifuge at 150,000g (35,300 rpm) for 3 h over the plant sucrose cushion buffer.

  • vii. Carefully wash or rinse the pellet with resuspension buffer for plant ribosomes and then resuspend in 200 µL of resuspension buffer for plant ribosomes.

    • The pellet suspension contains mainly polysomes and ribosome monomers. The plant polyribosomal fraction contains two types of ribosomes, cytoplasmic (80S) and chloroplast (70S). To include the free ribosomal subunits, increase the centrifugation time to at least 16 h.

  • viii. Use, store (at −80°C), or further purify the resuspended pellet as desired.

    • For further purification of the polysomes, proceed to Protocol: Purification of Polysomes (Rivera et al. 2014b).

  For Chloroplast Ribosomes

  • ix. Centrifuge at 55,000g (21,400 rpm) for 12 h over the high-salt sucrose cushion buffer for chloroplast ribosomes.

  • x. Remove the sucrose by rinsing the pellet in high-salt sucrose cushion buffer without sucrose, 1:1 (v:v) ratio, and quickly spin the resuspension at 10,000g (9100 rpm) for 10 min at 4°C.

  • xi. Use the ribosome-containing pellet or store at −80°C.