Using this protocol, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function. Purification of ubiquitinated proteins in mammalian cells in comparison with the purification in vitro retains the ubiquitin linkage mode of target proteins under more radiological condition. Begin by adding 1.5 milliliters of reduced serum medium in three 15 milliliter centrifuge tubes.
Add plasmid DNA ready for transfection to each tube and add 0.2 micrograms of green fluorescent protein plasmid to monitor the transfection efficiency. Add 78 microliters of liposome transfection reagent to 4.5 milliliters of reduced serum medium in another centrifuge tube to dilute the liposomes. Mix thoroughly by flicking the tube and then allow it to stand for five minutes at room temperature.
Add 1.5 milliliters of the diluted liposome solution into each tube containing 1.5 milliliters of the diluted DNA solution. Mix thoroughly and cross-Link the plasmids with the liposomes for at least 20 minutes at room temperature. Discard the original medium from the Petri dishes and add nine milliliters of the reduced serum medium into each dish.
Add three milliliters of the liposome DNA mixture and mix the solution by gently shaking the plate back and forth three times and then left and right three times for even distribution of the mixture on the plate. Culture the cells in the humidified incubator at 37 degrees Celsius with 5%carbon dioxide. Replace the medium after four to six hours and continue to culture the cells for 24 to 36 hours.
After 24 to 36 hours, treat the cells with MG132 at a final concentration of 10 micromolar for four to six hours. Discard the medium and wash the cells two times with ice cold PBS. Add one milliliter of PBS to the dish.
Remove the cells by scraping them with a clean scraper and transfer the cell suspension into microcentrifuge tubes. Centrifuge at 700 G for five minutes to collect the cell pellets. Add 800 microliters of the ice cold FLAG lysis buffer supplemented with protease inhibitor cocktail to the cells in each tube.
Mix the cells using a vortex oscillator or a pipette gun and then incubate the mixture on a rotator at four degrees Celsius for 30 minutes. Ultrasonicate the mixture on the ice with each pulse lasting one second, and subject the mixture to five to 10 brief pulses. Incubate the mixture on a rotator at 40 RPM for 30 minutes at four degrees Celsius.
Then centrifuge the ultrasonicated samples at 8, 000 G for 20 to 30 minutes at four degrees Celsius. Transfer the supernatant to a new microcentrifuge tube. Aliquot 80 microliters of the cell extract and mix with 20 microliters of 5X SDS loading buffer.
Boil the samples at 98 degrees Celsius for five minutes. Cool on ice for two minutes and store at minus 20 degrees Celsius until use. Use these samples as the input group to monitor protein expression.
Next, add 30 microliters of the anti-FLAG M2 antibody conjugated beads to the remaining cell extracts and incubate on a rotator at four degrees Celsius for at least four hours or overnight. Centrifuge at 1, 500 G for two minutes at four degrees Celsius to collect the beads. Add one milliliter of the ice cold FLAG lysis buffer to the beads and mix by inverting the tube several times.
Repeat this step four to six times, then add 40 microliters of the FLAG peptides at a final concentration of 200 nanograms per microliter to the beads. Incubate on a rotator for two hours as shown earlier and then centrifuge at the same temperature. Transfer the supernatant to a new microcentrifuge tube and add 10 microliters of 5X SDS loading buffer.
Boil the mixture at 98 degrees Celsius for five minutes and then cool it on ice for two minutes. Add one milliliter of the ice cold PBS to the cell pellets and mix evenly. Aliquot 100 microliters of the cell suspension into a microcentrifuge tube as the input sample and centrifuge at 700 G for five minutes at four degrees Celsius to collect the cell pellets.
Add 80 microliters of FLAG lysis buffer to the input sample and mix the cells using a vortex oscillator as shown earlier. Lyse the cells on ice for one hour and then centrifuge again for 20 to 30 minutes. After centrifusion aliquot 80 microliters of the supernatant into a new microcentrifuge tube and add 20 microliters of 5X SDS loading buffer to the supernatant.
Boil the mixture at 98 degrees Celsius for five to 10 minutes and then cool on ice for two minutes. Centrifuge the remaining 900 microliters of the cell suspension at 700 G for five minutes at four degrees Celsius to collect the cell pellet. Add one milliliter of ubiquitin buffer 1 to the cell pellets and mix by pipetting up and down several times to distribute the cells evenly.
Subject the cell lysates to 10 to 20 rounds of ultrasonication on ice until the solution is no longer viscous, and then centrifuge the solution. Transfer the supernatant to a new microcentrifuge tube and add 30 microliters of nickel charged resin to the supernatant. Incubate on a rotator at 15 RPM for four hours or overnight at room temperature and then centrifuge for two minutes.
After centrifusion, collect the beads and add one milliliter of ubiquitin buffer 1 to it. Incubate with rotation in a shaker for 10 minutes at room temperature and then centrifuge again at 1, 500 G for two minutes, as shown previously. Add one milliliter of ubiquitin buffer 2 to the beads and perform incubation followed by centrifugation, as shown previously, to collect the beads.
Repeat this step once. Next, add one milliliter of PBS to the beads and follow the same procedure for incubation and centrifugation to collect the beads. Repeat this step once again.
Elute bound proteins by incubating the beads with 40 microliters of imidazole at a final concentration of 0.5 molar for one hour at room temperature. After incubation, centrifuge again at 1, 500 G for two minutes. Transfer the supernatant to a clean microcentrifuge tube and add 10 microliters of 5X SDS loading buffer.
Boil the solution at 98 degrees Celsius for five minutes and then cool it on ice for two minutes. Resolve the samples by SDS-PAGE and then transfer them to a nitrocellulose membrane by western blotting to detect target proteins using the corresponding antibodies. Start the chemiluminescence imaging system to detect signals from western blotting.
Place the nitrocellulose membrane in the camera obscura face up. Encode the substrate solution evenly on the membrane using a pipette gun. Finally, select the automatic exposure procedure to capture the chemiluminescent signals and manually adjust the exposure time until an ideal signal is acquired.
Total p53 protein, including ubiquitinated p53, was immunoprecipitated with FLAG M2 beads from H1299 cells under non denaturing conditions. The eluate was subjected to western blotting with anti p53 and anti hemagglutinin or with anti p53 and monoclonal antibodies. Here, the crude cell extracts or input were subjected to western blotting with anti p53 and anti MDM2 monoclonal antibodies.
The signal from ubiquitinated p53 protein which appears as a smeared band from low to high molecular weight was markedly increased when MDM2 was ectopically expressed in these cells. This result suggests that the ubiquitinated p53 protein has been effectively purified from cells. Next, the cells were lysed under denaturing conditions.
The total cellular ubiquitinated proteins were pulled down with nickel charged resin and were then subjected to western blotting with anti p53 and anti hemagglutinin monoclonal antibodies. Ubiquitinated p53 protein was detected using an anti p53 antibody and an anti hemagglutinin antibody. Here at the crude cell extracts or input were subjected to western blotting with anti p53 and anti MDM2 monoclonal antibodies.
The results showed that the total ubiquitinated protein level was unchanged but the ubiquitinated p53 protein level was dramatically increased after MDM2 was overexpressed in these cells. This indicates that the total ubiquitin proteins containing ubiquitinated p53 were effectively pulled down from cell lysates under denaturing conditions. When attempting this procedure, remember to treat the cells with MG132 before cell collection and ultrasonicate the cells properly on ice for the purification of ubiquitinated proteins.
