4. Place the black latex cap on the tube, which will displace some
liquid from the top.
5. Repeat steps 1–4 for each additional tube corresponding to as
many ribosome samples as required.
6. Place the tubes in a gradient-forming tube holder such that
there are an even number of tubes, and their weight is counterbalanced. Place the holder on the Gradient Station, and run the
station using the preprogrammed settings for SW28 rotor
(10–40% sucrose gradient). It usually takes 6 min to complete
the process.
7. Place the tubes in a refrigerator for at least ~2 h for equilibrating the temperature of the sucrose gradient. This step is not
necessary if the gradient is made with ice-cold buffer.
3.6 Sucrose Gradient
Ultracentrifugation
and Fractionation
1. Load ~2.4 nanomoles of crude ribosomes (~OD 260 ¼ 100 U)
on the top of the sucrose gradient (see Note 11).
2. Place the tube in SW28 rotor such that each tube is precisely
counterbalanced either with another sample or with a blank
gradient.
3. Centrifuge the samples in an ultracentrifuge at 24,000 rpm
(~104,000 Â g, r max ) for 16 h at 4
C.
4. After centrifugation, carefully remove samples and place them
into the holster and transfer to 4
C until ready for fractionation (see Note 12).
5. Turn on the syringe pump and the fraction collector of Brandel
sucrose gradient fractionating system. It takes about 20 min for
the system to stabilize, until the green indicator light turns on.
6. Release the piston of the syringe pump from the glass syringe
using the toggle switch on the pump using the normal speed
settings.
7. Fill the glass syringe with 60% sucrose solution, and place it
back into the slot of the system. Engage the piston by moving
the toggle switch forward. Connect the syringe to the bottom
needle of the density gradient flow cell through a Teflon
tubing.
8. Connect the top of the flow cell to the fraction collector.
Ensure that optical filter for 280 nm is inserted in the UA-6
detector (see Note 13).
9. Place an empty ultracentrifuge tube in the flow cell and tighten
the bottom such that the needle pierces through the bottom of
the tube and reaches about half an inch into the tube.
10. Fill up the tube 60% sucrose from the syringe using the syringe
pump at manual high-speed (6.0) setting, until the fluid flows
through the top tubing into the detector of the fraction collector. Adjust the baseline of the detector to zero, with the
sensitivity setting of 2.0.
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Yunlong Li et al.
liquid from the top.
5. Repeat steps 1–4 for each additional tube corresponding to as
many ribosome samples as required.
6. Place the tubes in a gradient-forming tube holder such that
there are an even number of tubes, and their weight is counterbalanced. Place the holder on the Gradient Station, and run the
station using the preprogrammed settings for SW28 rotor
(10–40% sucrose gradient). It usually takes 6 min to complete
the process.
7. Place the tubes in a refrigerator for at least ~2 h for equilibrating the temperature of the sucrose gradient. This step is not
necessary if the gradient is made with ice-cold buffer.
3.6 Sucrose Gradient
Ultracentrifugation
and Fractionation
1. Load ~2.4 nanomoles of crude ribosomes (~OD 260 ¼ 100 U)
on the top of the sucrose gradient (see Note 11).
2. Place the tube in SW28 rotor such that each tube is precisely
counterbalanced either with another sample or with a blank
gradient.
3. Centrifuge the samples in an ultracentrifuge at 24,000 rpm
(~104,000 Â g, r max ) for 16 h at 4
C.
4. After centrifugation, carefully remove samples and place them
into the holster and transfer to 4
C until ready for fractionation (see Note 12).
5. Turn on the syringe pump and the fraction collector of Brandel
sucrose gradient fractionating system. It takes about 20 min for
the system to stabilize, until the green indicator light turns on.
6. Release the piston of the syringe pump from the glass syringe
using the toggle switch on the pump using the normal speed
settings.
7. Fill the glass syringe with 60% sucrose solution, and place it
back into the slot of the system. Engage the piston by moving
the toggle switch forward. Connect the syringe to the bottom
needle of the density gradient flow cell through a Teflon
tubing.
8. Connect the top of the flow cell to the fraction collector.
Ensure that optical filter for 280 nm is inserted in the UA-6
detector (see Note 13).
9. Place an empty ultracentrifuge tube in the flow cell and tighten
the bottom such that the needle pierces through the bottom of
the tube and reaches about half an inch into the tube.
10. Fill up the tube 60% sucrose from the syringe using the syringe
pump at manual high-speed (6.0) setting, until the fluid flows
through the top tubing into the detector of the fraction collector. Adjust the baseline of the detector to zero, with the
sensitivity setting of 2.0.
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Yunlong Li et al.