The overall goal of this procedure is to measure the effects of stress conditions on translation, initiation of mammalian cells and tissues by polysome profiling. This is accomplished by first treating mammalian cancer cells with an anti-cancer drug and then preparing cell extracts. The second step is to make sucrose, gradients.
Next, the cell extracts are loaded onto the sucrose, gradients, and ultra centrifuged to separate ribosomal fractions. Ultimately, western blot analysis using specific antibodies is performed to assess the association of tested proteins with polysome. The main advantage of this technique of a existing method like metabolic labeling, is that it allow us to assess the effect of stress conditions on the initiation step of translation.
This method can help answer key questions in stress therapeutic field, such as whether and how anti-cancer drugs affect translation initiation. The Implication of this technique extend toward cancer therapy because it helps to reveal new regulatory pathways of translation Initiation. Though this method can provide insight into translation initiation regulation in cell cultures, it can also be applied to other systems such as isolated tissues, including brain and tumors.
To prepare the automated density gradient fractionation system first, wash the tubing system of the apparatus with 0.1%SDS for five minutes. Then wash the tubing system with 70%ethanol for five minutes, followed by RNA Zap solution for five minutes. And lastly, with DEPC, water, dry the tubing system by pumping air into the system.
Next, prepare 15%and 55%Sucrose solutions generate the linear 15 to 55%sucrose gradient using an ISCO model one 60 gradient former. Following the manufacturer's instructions, it is important to carefully control the tris peristaltic pump speed in order to avoid creation of bubbles or turbulences in the gradients during the time when the gradient maker program is running cool the ultra centrifuge to four degrees Celsius. Keep the gradients at four degrees Celsius until use this experiment utilizes low passage helo cervical cancer cells previously plated to reach 80%confluence.
On the day of the experiment. It is important to make extractions from fresh cells and to avoid RNA contamination during this procedure, 90 minutes prior to adding the anti-cancer drug, replace the medium of the cells with fresh, complete medium. To stimulate translation, return the cells to the incubator.
Add the anti-cancer drug directly to the cell culture as a control add. DMSO incubate for four hours at 37 degrees Celsius, 5%carbon dioxide after four hours, place plates on ice and wash cells three times with cold PBS harvest cells in one milliliter of lysis buffer for best results. Use approximately 12 million cells for each experimental condition.
Transfer cells from scraped plates to an einor tube and mix well by passing them 15 times through a syringe. Let the cell lysates rest on ice for 15 minutes. Next, put a few drops of cell lysate onto a slide and assess cellular lysis by phase contrast microscopy using a 10 x objective.
Once cell lysis has been verified, clarify the cell lysate by centrifugation at 11, 000 Gs for 20 minutes at four degrees Celsius. Transfer the soluble lysate containing the poly ribosomes to a new tube and keep it on ice prior to loading the cytoplasmic extracts onto sucrose gradients. Measure the concentration of RNA present using a spectrophotometer carefully and slowly load approximately 20 OD two 60 units of the extract onto the 15 to 55%sucrose gradient.
Make sure that there is two to three millimeters of available space at the surface of the tube to avoid overflowing the tube during centrifugation. Load the tubes onto the rotor. Place the rotor onto the ultra centrifuge and centrifuge the gradients at 230, 000 Gs at four degrees Celsius for two hours and 30 minutes.
When centrifugation is complete, remove the tubes and place them on ice carefully without disturbing the gradients. To begin the fractionation of the cytoplasmic extracts, place the sucrose gradients on an automated density fractionation system and proceed with automated fractionation and collection of fractions as described by the manufacturer. Collect each 0.5 milliliter fraction into individual einor tubes with continuous monitoring of absorbance at 250 nanometers in parallel to the gradient fractionation operation, the poly ribosomal profile will be editing on the chart paper at the end of each run.
Transfer eend tubes with collected fractions onto ice. To begin this procedure, add two volumes of 100%cold ethanol to each collected fraction, and let the RNA protein complexes precipitate at minus 20 degrees Celsius overnight. For extraction of proteins, centrifuge each RNA protein precipitate at 11, 000 Gs for 20 minutes at four degrees Celsius after washing with 70%ethanol air dry and resuspend the precipitate.
An SDS page sample buffer Before proceeding with western blot analysis for extraction of RNA Resus, suspend each RNA protein precipitate in lysis buffer containing 0.1%SDS digest a protein component of the precipitate with two milligrams per milliliter proteinase K for 30 minutes. In a 55%Celsius water bath, add one volume of phenol chloroform, two volumes of chloroform and 0.1 volume of two molar sodium acetate pH four. Let it rest on ice for 15 minutes.
Centrifuge at 10, 000 GS at four degrees Celsius for 20 minutes. Collect the resulting aqueous phase of each sample precipitate RNA by adding one volume of isopropanol and 0.2 micrograms per microliter of glycogen and leaving it at minus 20 degrees Celsius overnight. On the following day, spin the precipitate at 10, 000 Gs at four degrees Celsius for one hour.
Remove the supernatant and wash the RNA pellet with 70%cold ethanol resuspend the RNA pellet into a small volume of RNAs. Free water. Assess the quantity and quality of RNA using the spectro photometer.
Shown here are representative polysome profiles from cytoplasmic extracts. Prepared from helo cells grown in either normal conditions or after treatment with the proteasome inhibitor for four hours and centrifuge done a 15 to 55%volume to weight linear sucrose gradient. The positions of 40 s ribosomes 60 s, ribosomes 80 s, monos and polysome are indicated in each profile.
Fractions from the top to the bottom of the gradient are shown from left to right fractions were collected and analyzed by Western blot with antibodies against the large ribosomal L 28 protein, as well as the polysome associated protein FMRP that serve as controls for polysome integrity under normal growth conditions. Translation initiation complexes are converted into poly ribosomes whose detection by polysome profiling atest for an active translation initiation as observed here. Treatment with the proteasome inhibitor reduces polysome peaks with a concomitant increase in a DS Monos indicating an inhibition of translation initiation While attending this procedure, it's important to work in an r nase free environment to handle gradients carefully and to control for cell lysis Following this procedure.
Other methods such as western blood and north blood can be performed in order to answer additional questions like with association of specific protein or M MNA with polysome is affected by anti-cancer treatment After its development. This technique paved the way for researchers in the field of ary translation to explore the impact of regulating translation initiation factors in human diseases such as cancer and neurodegenerative disease.
