This protocol describes how to generate a polysome profile which reveals molecular details about the activity of ribosomes inside the cell. This technique allows users to obtain the valuable information provided by a polysome profile, who do not have access to automated gradient fractionation systems. Take your time while preparing the gradients, and store the gradients in a location where they will not be disrupted by a compressor or other mechanical operations as they settle into a linear gradient.
To begin, prepare stock solutions of 7 and 47%sucrose in sucrose gradient buffer. Filter sterilize the sucrose stock solutions through a 0.22 micron filter. Prepare 14 milliliters of 17, 27, and 37%sucrose solutions by dispensing and mixing the 7 and 47%stock solutions.
Place six polypropylene centrifuge tubes into a full view test tube rack. Ensure that there is enough space between the tubes so that the actions with one tube do not disturb the others. Attach a nine inch, 22 gauge needle with a blunt tip to a three milliliter syringe, and perform a test fill and dispense to ensure that the syringe can hold the sucrose solution without any dripping before setting up the gradients.
Add two milliliters of the 7%sucrose to the bottom of each centrifuge tube. Then, add two milliliters of the 17%sucrose beneath the 7%solution, by positioning the needle tip within the immediate vicinity of the tube bottom. Carefully and slowly dispense the solution.
Repeat the procedure with two milliliters each of the 27, 37 and 47%sucrose solution, ensuring that each layer is distinguishable from the other by a line marking the separation of densities. Store the gradients at four degree Celsius overnight to allow them to settle into a continuous, increasing percentage of sucrose. After the growth and harvesting of yeast, Saccharomyces cerevisiae cells, resuspend the cells in chilled 700 microliters of polysome extraction buffer.
Add 100 units of RNAse Inhibitor. Then, add 400 microliters of pre-chilled glass beads with an approximate size of 425 to 600 microns. Transfer the mixture to a 1.5 milliliter centrifuge tube with the glass beads, and disrupt the yeast cells by vigorous agitation in a bead-beater for five minutes.
After disrupting the cells, clarify the lysate by centrifugation. Determine the concentration of the RNA in the clarified lysate by measuring the absorbance at 260 nanometers, using a fluorescence-based RNA detection system. Ensure that RNA concentration is 0.5 to one microgram per microliter.
If the RNA concentration is too low, reduce the volume of extraction buffer used to resuspend the cells. Carefully load the lysate onto the top of the gradients. Place the pipette tip against the inner wall at the top of the polypropylene tube.
Angle the tube and slowly dispense the lysate onto the top of the gradient, dribbling against the wall. Gently place the tubes into the pre-chilled buckets of a swinging bucket rotor, and centrifuge the gradients. After centrifugation, carefully removed the centrifuge tubes from the swinging bucket rotor, and place them in a tube holder.
Label the 96-well plates to store the fractions and pre-chill on ice. Collect 100 or 200 microliter fractions starting from the top of the gradient, by carefully inserting a pipette tip into the top of the gradient, and collecting the fractions until the entire gradient is aliquoted. Measure the absorbance of each fraction at 254 nanometers with a spectrophotometer against the 7 and 47%sucrose solutions as blanks.
Create the polysome profile by plotting the fraction number versus absorbance. Three representative polysome profiles from yeast S.Cerevisiae, are shown. The crest of each ribosomal subunit and polysome peak is apparent on each profile.
A representative profile from an automated density fractionation system is shown here. This profile was produced from a continuous absorbance profile as the sucrose gradient was displaced from the bottom up by a chase solution, through a detector flow cell and collected in fractions. The polysome profile generated by hand fractionating 200 and 100 microliter samples is shown here.
The most important thing to remember with this procedure is to not disrupt the gradients. Take care not to introduce air bubbles or disrupt the gradient by dispensing solution too rapidly. Following this procedure, RNA can be extracted from the gradients for further analysis to determine mRNAs that are associating with active ribosomes
