This protocol is for polysome fractionation by sucrose gradient, to view
translational status of cells and ribosome-association of mRNAs and
proteins, using the BioComp gradient station
(http://www.biocompinstruments.com/). It’s based on the BioComp
instructions, with help from Wesley Clark.
Read the BioComp instruction book for more details of gradient station
operation, especially for troubleshooting.
Components
Wet ingredients
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Sample: 100-500 ul cell lysate, with assembled
polysomes, at OD260 ~ 100.
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Mammalian Polysome gradient buffer: 100mM KCl, 7.5mM MgCl2, 5mM
Tris-HCl pH7.5, or,
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Yeast Polysome gradient buffer: 140mM KCl, 5mM MgCl2, 5mM Tris-HCl pH7.5
(NOTE: Can adjust salt, Mg, pH, etc; common additions are cycloheximide to
freeze ribosomes in place, heparin to reduce nonspecific RNA-protein binding and
inhibit RNase, DTT as reducing agent, RNase inhibitors. I’ve made good
polysome gradients without any of these at 2-10mM MgCl2, pH 6.5-7.5. Degraded
DTT is really bad as it absorbs over the RNA spectrum.)
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50% Sucrose buffer: 0.5g/ml sucrose in polysome gradient buffer, filter sterilized.
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10% Sucrose buffer: 0.1g/ml sucrose in polysome gradient buffer, filter sterilized.
Dry ingredients
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BioComp Instruments gradient station.
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Beckman Coulter S-rated ultracentrifuge, e.g. Optima XP-100.
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Beckman SW28 rotor and SW28.1 (or SW28) tube buckets, kept in cold room. Rotor
must be placed only on rotor-holding platform, do not scratch speed ring on bottom.
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Seton open-top polyclear centrifuge tubes SW28.1 (part.no 7042). Alternatively,
SW28 (part.no 7052); SW28 are wider and slightly shorter.
Prepare gradients
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Layer sucrose into centrifuge tube: place a polyclear centrifuge tube in the
marker block. Pour 10% sucrose buffer into the tube up to the top of the marker
block (roughly 10ml for SW28.1 / 19.5ml for SW28). Load 50% sucrose buffer into
a 50ml Luer-lok syringe with a layering cannula (BioComp 106-211) attached; with
the needle pointing upwards, expel air from the needle. Quickly and smoothly
invert the syringe so the needle is in the bottom centre of the half-filled
centrifuge tube. Slowly layer 50% sucrose buffer at the bottom of the tube until
the 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 the
tube, ensuring tube is sealed round edges of cap. Repeat for desired number of
tubes (2, 4 or 6 total), and place filled tubes in magnetic tube rack.
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Level the gradient maker platform on the gradient station: turn
gradient-maker on, choose GMST menu option. The machine will prompt you to level
the gradient-making platform. Place a bubble level on the platform with axis
perpendicular to the side plate of the machine and use the UP and DOWN menu
options to level the platform (the machine should already be level front to
back); press DONE.
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Make gradients: place magnetic tube rack on gradient-making platform. Select
GRAD option to arrive at gradient menu. The first time, go to LIST and choose
the 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 gradient
immediately previously, simply select LAST from the gradient menu. Press USE to
start 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 to
disturb the gradient. Remove capillary cap from tube and, using long-nosed
pliers if necessary, place in rotor bucket in bucket rack.
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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 tube
as necessary to ensure paired tubes are within 0.1g in mass.
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Prepared gradients can be stored at 4C for a day or two.
Spin
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Load sample and assemble rotor: take sample, SW28.1 (or SW28) rotor,
bucket rack, and 200 ul pipette to ultracentrifuge. Load sample in
each tube by placing filled pipette tip in meniscus at side of tube,
and pipetting slowly; you should see the sample spreading out across
top of liquid. Gently place lid on tube and screw cap on bucket.
Hang buckets in numbered slots on rotor, checking that both hooks
are attached for every bucket.
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Set up centrifuge: turn on centrifuge, break vacuum and open
spin chamber. Select 27500 rpm, 3.5hrs, 4C, with vacuum, on
centrifuge controls. Place rotor assembly on axle, and seal
spin chamber. Start centrifuge and fill out centrifuge logbook.
Check on the centrifuge after 15 minutes to ensure it is
running smoothly.
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After running for 3.5hrs, the centrifuge takes several minutes
to brake. Once the centrifuge has stopped spinning, release the
vacuum, carefully remove rotor from spin chamber and place buckets
in bucket rack.
Set up gradient station
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Take bucket rack with gradients to gradient station, very gently.
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Turn on the gradient station, the UV monitor, the fraction collector,
and the linked computer. Connect the USB cable, start the gradient profiler
program on the computer and enter appropriate parameters.
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Flush the line and calibrate the UV monitor: press DRAIN on fraction
collector. On the gradient station, from the initial screen press FRAC, then
FRAC. Hold RINSE on gradient station to flush the line. Half-fill a centrifuge
tube with 10% sucrose buffer, turn front dial so that vacuum plunger descends at
about 0.5 mm/s. Once plunger is a few cm into liquid and drips are coming out of
the fraction collector, press AUTO ZERO on the UV monitor.
Collect fractions
For each ultracentrifuge tube with sample:
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Initialize fraction collector: ensure there are 30x clean 1.5ml
tubes in the two middle rows of the tube holder. Pre-label the tubes
if desired. Press END, then START, and make sure drip outlet is
above tube 0.
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Remove bucket cap, remove centrifuge from bucket using long-nosed
pliers, and attach locking top to centrifuge tube. Place tube in
tube holder on gradient station, locking the tube in place by
rotating the cap to lock in place. Slide tube holder onto mount on
top of gradient station with window facing to the right, and turn
clockwise so window is facing towards you.
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On gradient station, press FRAC once or twice to get to the fractionation
menu, 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 full
counterclockwise position. Move plunger downwards by turning dial to the right
until speed = 1.0 mm/s; move plunger slowly (0.2 mm/s) as it approaches the
gradient surface, so that you can stop (by turning dial fully left) as soon as
the plunger has sealed on the gradient. Press RESET on gradient station. In
subsequent tubes from same run, reset to same position – i.e. 0.0 mm on display
– from previous tube.
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On the gradient profiler program on the computer, press RECORD. Then
press START on gradient station.
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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 output
on the computer, and press NEW RUN to record the next gradient.
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Note that the first 1-3 tubes in the fraction collector are usually
empty, so that the tube number on the rack is offset from
that 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
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Check all equipment for potential sucrose gradient spills and
clean thoroughly with damp cloth and dilute ethanol; this is a very
sticky spill. In particular, clean the plunger and flush the tubing
with water.
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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).
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Store rotor and buckets in cold room, on rotor holding platform.