The overall goal of this procedure is to separate and compare the mono zone and poly ribosome fractions from cell lysates. This is accomplished by first culturing the cells of interest. The second step of the procedure is to prepare the cell lysate.
The third step of the procedure is to centrifuge the lysate through a sucrose gradient. The final step of the procedure is to fractionate the gradient and visualize the different ribosomal populations through a UV trace. Ultimately, results can be obtained that show changes in translation under different experimental conditions through poly ribosome profile analysis.
Hi, I am Anthony Esposito from the lab of Dr.Terry Goss Kinsey in the Molecular Genetics, microbiology and immunology department at UMD and J Robert Wood Johnson Medical School. And I'm Marcus Lewis. Also from McKinsey Lab here at R-W-J-M-S.
Today we're gonna show you a procedure for poly ribosome analysis. We use this procedure in our lab to study alterations and protein synthesis that arise from mutations or environmental factors. So let's get started.Sucrose.
Gradients should be prepared one day before use to allow gradients to become continuous. Mix the appropriate volumes of 7%and 47%sucrose stock solutions to make 48 milliliters of each of the following sucrose concentrations, 7%17%27%37%and 47%Add one molar DTT to each sucrose solution to a final concentration of one millimolar. In order to pour the gradients, attach a pasta pipette to a 20 milliliter syringe by securing and sealing with para film.
Add seven milliliters of 7%sucrose to the bottom of a one inch by 3.5 inch poly aamer ultra centrifuge tube. Next, add seven milliliters of 17%sucrose solution under the 7%solution by placing the pasta pipette tip near the bottom of the tube pipette slowly. It should take no less than 20 seconds to pour each layer.
Repeat with seven milliliters each of 27%37%and 47%sucrose solutions when the gradient has been bored. You should observe clear lines between the layers indicating that minimal mixing has occurred. Store all gradients of four degrees Celsius overnight, along with the rotors, bottles and tubes that will be used to harvest samples.
To prepare extracts from yeast cells, start by growing hundred 25 milliliters of yeast culture to an OD 600 of 0.8 to one. Add cyclo heide to the culture to a final concentration of 100 micrograms per milliliter and continue shaking at 30 degrees Celsius for 15 minutes. After 15 minutes, pour the culture into a 250 milliliter centrifuge bottle and fill with ice.
Keep everything on ice from this point onward centrifuge at 8, 000 times gravity for five minutes at four degrees Celsius. Resus, suspend the pellet in 10 milliliters of ice, cold lysis, buffer, and transfer to 15 milliliter polypropylene tubes. Centrifuge at 5, 900 times gravity for three minutes of four degrees Celsius Resus.
Suspend the pellet in 0.5 milliliters of lysis buffer. Transfer the suspension to a 1.5 milliliter micro fuge tube and then add 0.5 milliliters of chilled baked acid washed glass beads. Vortex the cells for four 32nd intervals, calling the tube on ice for 30 seconds between each interval.
Then add another 0.5 milliliters of lysis buffer into the 1.5 milliliter tube centrifuge at 5, 000 times. Gravity for five minutes at four degrees Celsius. Transfer the supernatant to a new 1.5 milliliter micro centrifuge tube and centrifuge at maximum speed for 10 minutes at four degrees Celsius.
Lastly, transfer the supinate to a new 1.5 milliliter micro centri tube. The extract can be stored at minus 80 degrees Celsius to transfer the lysate to the sucrose gradient. Place the tip of the pipette against the wall of the tube near the surface of the gradient and gently layer the lysate on top of the gradient.
For a 35 milliliter gradient, 500 microliters of lysate is typically loaded. Using forceps, carefully lower the gradient into the bucket of a swinging bucket rotor. Centrifuge the gradients at 100, 000 times gravity for four hours at four degrees Celsius.
Approximately 30 minutes prior to the completion of the four hour spin. Attach the needle to the tube piercer and attach the pen to the online absorbance fluorescence monitor. Turn on the power for the absorbance monitor to allow a stable baseline to be reached prior to analyzing samples.
Electronic acquisition of the PolyZone profile can easily be obtained in real time by attaching a DI 1 48 U data acquisition unit. To the chart recorder, fill the syringe pump with 50 milliliters of florin nuts using the reverse flow rapid setting. Make sure that there are no air bubbles present.
Connect the hose from the syringe to the tube piercer. And briefly turn on the flow of Flo inert at six milliliters per minute in the forward direction. To clear the hose of any air bubbles, place a series of tubes under the hose at the end of the flow cell to collect sample runoff.
Prepare an ice bucket with a preformed well for holding the centrifuge tube containing the sucrose gradient. When the four hour centrifugation is complete, carefully remove the centrifuge tubes from the centrifuge rotor and place them on ice. Taking care to avoid disturbing the gradients.
To analyze cell extracts, place the gradient tube on the tube piercer and connect the top of the tube to the outlet and secure. Raise the bottom stage to the centrifuge tube so that the needle pierces the bottom of the tube. Begin the flow of Florin alert in the forward direction at six milliliters per minute.
Once the flut has begun flowing into the tube, monitor the movement of the needle on the online UV absorbance monitor. Once the needle begins to move, turn on the chart movement to a speed setting of 60. When the end of the poly ribosome profile has been reached, turn off the flow of Flo inert to the centrifuge tube and stop the chart movement at the absorbance fluorescence monitor.
Retract the Flo inert from the centrifuge tube into the syringe by beginning the flow in the reverse direction at a rapid rate, making sure not to draw sucrose from the tube into the syringe. Unscrew the centrifuge tube from the tube piercer, lower the needle and remove the tube. A representative trace of a ribosome extract prepared from yeast in the presence of cyclo heide is shown here.
UV absorbance peaks containing polyribosomes and A TS 60 s and 40 s ribosomes are indicated. We've just shown you how to perform poly ribosome analysis When doing this procedure. It's important to remember to work quickly, keeping samples cold and RNAs free.
So that's it. Thanks for watching and good luck on your experiments.
