Sucrose gradient protocol for polysome profiles (2023)

This protocol is for polysome fractionation by sucrose gradient, to viewtranslational status of cells and ribosome-association of mRNAs andproteins, using the BioComp gradient station(http://www.biocompinstruments.com/). It’s based on the BioCompinstructions, with help from Wesley Clark.

Read the BioComp instruction book for more details of gradient station operation, especially for troubleshooting.

Components

Wet ingredients

• Sample: 100-500 ul cell lysate, with assembledpolysomes, at OD260 ~ 100.

• Mammalian Polysome gradient buffer: 100mM KCl, 7.5mM MgCl2, 5mMTris-HCl pH7.5, or,

• Yeast Polysome gradient buffer: 140mM KCl, 5mM MgCl2, 5mM Tris-HCl pH7.5(NOTE: Can adjust salt, Mg, pH, etc; common additions are cycloheximide tofreeze ribosomes in place, heparin to reduce nonspecific RNA-protein binding andinhibit RNase, DTT as reducing agent, RNase inhibitors. I’ve made goodpolysome gradients without any of these at 2-10mM MgCl2, pH 6.5-7.5. DegradedDTT is really bad as it absorbs over the RNA spectrum.)

• 50% Sucrose buffer: 0.5g/ml sucrose in polysome gradient buffer, filter sterilized.

  (Video) Polysomal fractionation using sucrose gradients

• 10% Sucrose buffer: 0.1g/ml sucrose in polysome gradient buffer, filter sterilized.

Dry ingredients

• BioComp Instruments gradient station.

• Beckman Coulter S-rated ultracentrifuge, e.g. Optima XP-100.

• Beckman SW28 rotor and SW28.1 (or SW28) tube buckets, kept in cold room. Rotormust be placed only on rotor-holding platform, do not scratch speed ring on bottom.

• Seton open-top polyclear centrifuge tubes SW28.1 (part.no 7042). Alternatively, SW28 (part.no 7052); SW28 are wider and slightly shorter.

Prepare gradients

1. Layer sucrose into centrifuge tube: place a polyclear centrifuge tube in themarker block. Pour 10% sucrose buffer into the tube up to the top of the markerblock (roughly 10ml for SW28.1 / 19.5ml for SW28). Load 50% sucrose buffer intoa 50ml Luer-lok syringe with a layering cannula (BioComp 106-211) attached; withthe needle pointing upwards, expel air from the needle. Quickly and smoothlyinvert the syringe so the needle is in the bottom centre of the half-filledcentrifuge tube. Slowly layer 50% sucrose buffer at the bottom of the tube untilthe top of the meniscus is at the top of the tube (roughly 10ml; 19ml for SW28),and carefully remove needle from tube. Place the capillary cap on top of thetube, ensuring tube is sealed round edges of cap. Repeat for desired number oftubes (2, 4 or 6 total), and place filled tubes in magnetic tube rack.

   (Video) Polysome Profiling

2. Level the gradient maker platform on the gradient station: turngradient-maker on, choose GMST menu option. The machine will prompt you to levelthe gradient-making platform. Place a bubble level on the platform with axisperpendicular to the side plate of the machine and use the UP and DOWN menuoptions to level the platform (the machine should already be level front toback); press DONE.

3. Make gradients: place magnetic tube rack on gradient-making platform. SelectGRAD option to arrive at gradient menu. The first time, go to LIST and choosethe SW28.1 (or SW28) rotor option. Press DOWN until arriving at the 10-50% sucrose gradient option, and press USE. If the machine was used for 10-50% sucrose gradientimmediately previously, simply select LAST from the gradient menu. Press USE tostart making gradients, which takes roughly 10 minutes. From this point onwards,keep the tubes upright and make no sudden movements with them, so as not todisturb the gradient. Remove capillary cap from tube and, using long-nosedpliers if necessary, place in rotor bucket in bucket rack.

4. Balance the tubes: balance tube 1 with tube 4, 2 with 5, and 3 with 6.Placing bucket-tube assembly on scale, remove sucrose gradient from top of tubeas necessary to ensure paired tubes are within 0.1g in mass.

5. Prepared gradients can be stored at 4C for a day or two.

Spin

1. Load sample and assemble rotor: take sample, SW28.1 (or SW28) rotor, bucket rack, and 200 ul pipette to ultracentrifuge. Load sample ineach tube by placing filled pipette tip in meniscus at side of tube,and pipetting slowly; you should see the sample spreading out acrosstop of liquid. Gently place lid on tube and screw cap on bucket.Hang buckets in numbered slots on rotor, checking that both hooksare attached for every bucket.

2. Set up centrifuge: turn on centrifuge, break vacuum and openspin chamber. Select 27500 rpm, 3.5hrs, 4C, with vacuum, oncentrifuge controls. Place rotor assembly on axle, and sealspin chamber. Start centrifuge and fill out centrifuge logbook.Check on the centrifuge after 15 minutes to ensure it isrunning smoothly.

   (Video) Sucrose Density Gradient Centrifugation |

3. After running for 3.5hrs, the centrifuge takes several minutesto brake. Once the centrifuge has stopped spinning, release thevacuum, carefully remove rotor from spin chamber and place bucketsin bucket rack.

Set up gradient station

1. Take bucket rack with gradients to gradient station, very gently.

2. Turn on the gradient station, the UV monitor, the fraction collector, and the linked computer. Connect the USB cable, start the gradient profilerprogram on the computer and enter appropriate parameters.

3. Flush the line and calibrate the UV monitor: press DRAIN on fractioncollector. On the gradient station, from the initial screen press FRAC, thenFRAC. Hold RINSE on gradient station to flush the line. Half-fill a centrifugetube with 10% sucrose buffer, turn front dial so that vacuum plunger descends atabout 0.5 mm/s. Once plunger is a few cm into liquid and drips are coming out ofthe fraction collector, press AUTO ZERO on the UV monitor.

Collect fractions

For each ultracentrifuge tube with sample:

1. Initialize fraction collector: ensure there are 30x clean 1.5mltubes in the two middle rows of the tube holder. Pre-label the tubesif desired. Press END, then START, and make sure drip outlet isabove tube 0.

   (Video) Assessment Of Selective mRNA Translation In Mammalian Cells By Polysome Profiling l Protocol Preview

2. Remove bucket cap, remove centrifuge from bucket using long-nosedpliers, and attach locking top to centrifuge tube. Place tube intube holder on gradient station, locking the tube in place byrotating the cap to lock in place. Slide tube holder onto mount ontop of gradient station with window facing to the right, and turnclockwise so window is facing towards you.

3. On gradient station, press FRAC once or twice to get to the fractionationmenu, then SNGL for single run, and set the parameters to speed = 0.3, distance= 3.2 (for SW28.1; 2.6 for SW28), and 31 fractions. Rotate front dial to fullcounterclockwise position. Move plunger downwards by turning dial to the rightuntil speed = 1.0 mm/s; move plunger slowly (0.2 mm/s) as it approaches thegradient surface, so that you can stop (by turning dial fully left) as soon asthe plunger has sealed on the gradient. Press RESET on gradient station. Insubsequent tubes from same run, reset to same position – i.e. 0.0 mm on display – from previous tube.

4. On the gradient profiler program on the computer, press RECORD. Thenpress START on gradient station.

5. When finished, remove tubes from fraction collector and label them.Press EXIT 3 times on gradient station to return to fraction menu,and remove centrifuge tube from holder. Crucially, save the outputon the computer, and press NEW RUN to record the next gradient.

6. Note that the first 1-3 tubes in the fraction collector are usuallyempty, so that the tube number on the rack is offset fromthat reported in the gradient profiler software (BioComp instructions suggest this is solvable: good luck!). Tube 0 according to gradient profiler software is the first filled tube on the fraction collector. It may be easiest to label the tubes accordingly, after the fractions are collected.

Cleanup

1. Check all equipment for potential sucrose gradient spills andclean thoroughly with damp cloth and dilute ethanol; this is a very sticky spill. In particular, clean the plunger and flush the tubing with water.

   (Video) Eukaryotic Polyribosome Profile Analysis l Protocol Preview

2. Flush tubing with a full tube of 50% Ethanol to disinfect (DRAIN on fraction collector, from FRAC screen on gradient station lower plunger at about 0.5 mm/s).

3. Store rotor and buckets in cold room, on rotor holding platform.