Hi, I'm Chu from the laboratory of Jo at the Birmingham Institute for Medical Research. Today we will show you a procedure for how to detect protein new uation. We use this procedure in our laboratory to study parking or UBI three ligates and it substrate.
So let's get started. To begin this protocol, transfect cells with plasmids expressing both the target protein and an epitope tagged version of ubiquitin. Let the cells grow for about 40 hours, then move to the laboratory bench to lice the cells at the bench, aspirate off the liquid medium and wash the cells twice with PBS.
Next ly the cells by adding 100 microliters cell lysis buffer, which contains 2%SDS per six centimeter plate. Swirl each plate so that the buffer completely covers the cells. Next, use a cell scraper to collect the lysed cells and transfer them to a 1.5 milliliter eend tube.
Immediately place the tube onto a heat block and boil the lys cells for 10 minutes to further denature and dissolve proteins. After boiling, use a sonication device to shear the cells and their DNA. This step makes the cell lysates less viscous.
After sonication, add 900 microliters of dilution buffer and leave the samples to rotate at four degrees Celsius for 30 to 60 minutes. After the incubation centrifuge the diluted samples at 20, 000 Gs for 30 minutes at four degrees Celsius in order to obtain soluble proteins. Then careful not to disturb the pellet, transfer the supernatant to a new einor.
Lastly, measure the protein concentration in the supernatant by a method such as the modified Lowry assay we are using here And now. You are ready to use am immunoprecipitation to isolate your protein of interest from the others in the supernatant. The first step in immunoprecipitation is to prepare the beads conjugated to antibodies against your protein of interest.
You can use commercially available antibody conjugated beads or make your own immobilized antibody using cross linkers. As we have here, prepare the immobilized antibody in a compatible buffer to make a 50%slurry to set up the amino precipitation reaction. Cut the narrow end of a P 200 pipette tip and transfer 14 to 20 microliters of resin to 500 to 1500 micrograms of prepared cell lysates.
Adjust the amount of cell lysate you use according to the transfection efficiency of your cells. Leave the cell lysate bead mixture to rotate at four degrees Celsius overnight. During this time, the bead conjugated antibody will specifically bind your target protein.
The next day, spin down the beads at 5, 000 Gs for five minutes at four degrees Celsius to pellet your protein. Now bound to the beads, aspirate the supernatant and wash the resin twice with the high salt washing buffer by spinning at 5, 000 Gs for five minutes. At four degrees Celsius, spin the beads for a final time at 20, 000 GS for 30 seconds and aspirate the residual washing buffer.
Now that you have purified your protein of interest, it is time to run out the protein on an SDS page Gel. First, boil the bead resin with two times SDS loading buffer to denature the proteins and give them a negative charge. Load your prepared samples onto an SDS page.
Gel L transfer the proteins to a membrane using your laboratory's protocol for western blotting. Then pro the blot with antibodies against ubiquitin or the target protein and develop the membrane on film to visualize ubiquitinated proteins. We have just shown you how to detect protein ubiquitination in cultured cells.
This same experiment can be performed in vitro in a test tube. This reaction has a total volume of 40 and can be done in a PCR tube. Prepare each reaction as follows.
First, add five times UBIQUITINATION buffer, which contains a TP and reducing agent DIO three etol, DTT. Next, add the ubiquitination enzymes E one and E two. Add the substrates, ubiquitin, and target protein.
Bring the total reaction volume to 40 microliters with water as controls. Prepare similar reactions in the absence of either ubiquitin E one, E two, or the target protein. Incubate the mixture at 37 degrees Celsius for one hour or longer.
Stop the reaction by adding SDS page, sample buffer. Boil the sample for 10 minutes. Load the samples onto an SDS page gel and proceed with immuno blotting analysis as you did for the in vivo experiment.
Here is an example outcome of this protocol.Parkinson. A Parkinson's disease associated protein is the protein of interest here. And lane four shows ubiquitinated parin.
The typical strong smears or ladders are shown above the molecular weight of the unmodified parin to compare several experimental conditions, the cellular expression levels of the unmodified target protein, parin and ubiquitin that each condition also should be considered. This is an example of in vitro parin ubiquitination affinity purified parin was assay for in vitro ubiquitination in the presence of recombinant E one, E two and biotin tag ubiquitin biotin ubiquitinated parin was detected by streptavidin HRP. The ubiquitinated Parin appears as ladders above the molecular weight of the unmodified parin lane one, two, and four are controls which do not contain ubiquitin E one, E two, and parkin respectively.
We, we've just shown you how to detect protein new vaccination in the cultural cells. When doing this procedure, it's important to remember to prepare cell lysate in a highly tering condition and to dilute the cell lysate before adding the mobilized antibodies. So that's it.
Thank you for watching and good luck with your experiment.
