The overall goal of this experiment is to detect the Rab10 phosphorylation by LRRK2 using SDS-PAGE with a phosphate-binding tag. This method can help answer key questions in the Parkinson research field such as how disease mutations in LRRK2 affect Rab10 phosphorylation. The main advantage of this technique is that one can have a rough estimation of the stoichiometry of Rab10 phosphorylation using a simple western blot.
Many of the steps of this protocol are fairly standard and are described in sufficient detail by the text protocol accompanying this video. Here, we want to show you the steps that benefit most from visual instruction. Prepare plates of transfected HEK293 cells as described in the text protocol.
With the plates on ice, aspirate and discard the media. Next, carefully add two milliliters of DPBS to the corner of each plate and rock the plates to spread the solution. Then remove and discard the DPBS.
Next, to each plate add 100 microliters of lysis buffer. Tilt the plate and use a scraper to spread the buffer and release the cell lysate. Then aspirate the lysates and transfer them to microfuge tubes prechilled on ice and wait 10 minutes.
Next, collect debris-free cell lysates by centrifuging the tubes and transferring the supernatants to new tubes on ice. Then measure the protein concentrations of the cleared lysates using the Bradford assay. For the concentration data, prepare 100 microliter samples of the lysates for the gel analysis.
Prepare denser lysates to measure endogenous Rab10 compared to overexpressed hemagglutinin Rab10. Finish the preparation of the samples by incubating them at 100 degrees Celsius for five minutes. Then store the samples below minus 20 degrees Celsius until needed for up to six months or longer.
To begin, prepare the SDS-PAGE cells according to the text protocol. Then load the gel into the tank and secure it with binder clips. Next, fill the tank with running buffer and remove the air bubbles in the wells and under the gel using a needle attached to a syringe.
Now add manganese chloride to the samples and spin them down to remove the precipitates. Then load the gel with equal volumes of sample, 10 micrograms of protein to analyze overexpressed hemagglutinin Rab10 or 30 micrograms of protein to analyze endogenous Rab10. Load the empty lanes with sample buffer containing manganese chloride.
Also supplement the molecular weight marker with buffer and manganese chloride to make it the same volume as the samples. Now start running the gel at 50 volts. After the sample stack, increase the voltage to 120 to get separation.
When the dye front reaches the bottom of the gels, kill the voltage. Next, wash the gel three times as follows. Load the separation gel into a bath of clean transfer buffer with added EDTA and SDS.
Then let it rock on a shaker for 10 minutes at room temperature. After three washes, perform one more wash using transfer buffer with added SDS but without EDTA. Then transfer the gel using a wet tank.
Load the tank into a foam box filled with ice cold water, connect the tank to a power supply and start the transfer. A long transfer with efficient cooling is essential to the procedure. After the transfer, check the transfer by staining the membrane with Ponceau S, followed by a wash in TBST according to the text protocol.
Next, apply a block. Immerse the blot in milky TBST and rock the blot on a shaker for an hour at room temperature. Then remove and discard the blocking solution and apply the primary antibody solution.
Now rock the membrane overnight at four degrees Celsius. The next day, remove the primary antibody solution. Then wash the membrane with fresh TBST three times as before.
After the washes, immerse the blot in secondary antibody solution and incubate the membrane on a rocker for about an hour at room temperature. After the incubation, wash the membrane with TBST three times once again. Now develop the membrane.
Add one milliliter of ECL solution to a large sheet of plastic wrap. Then lay the membrane into the ECL and quickly flip it over to wet both sides. Next, lift the membrane and let it drain.
After draining, let one edge touch a paper towel for about five seconds to help wick off excess solution. Then wrap the membrane in stiff clear film without wrinkles and transfer it to a black tray for imaging. HEK293 cells were transfected with hemagglutinin Rab10 wild-type and 3xFLAG-LRRK2.
Wild-type kinase inactive in familial Parkinson disease mutations of LRRK2 were tested. Co-overexpression of LRRK2 and Rab10 resulted in a major band shift in the P-tag gel. In contrast, co-expression with a kinase inactive mutant resulted in no change in Rab10 mobility, implicating the kinase as the cause of the band shift.
Also as expected, the familial PD mutations all increased the band shift. The P-tag gel system was also used to analyze mouse 3T3-Swiss albino embryonic fibroblast cells treated with a LRRK2 inhibitor. The band shift corresponding to phosphorylated endogenous Rab10 disappeared when the cells were treated with GSK2578215A.
The same test was also performed using human A549 cells. In these experiments, the efficacy of the LRRK2 inhibitor was validated using the anti-p serine 935 LRRK2 antibody which is a well-established marker of LRRK2 inhibition. Once mastered, this technique only needs additional 14 minutes compared to a regular western blotting procedure which is for washing gels before transfer.
Following this procedure, other methods like mass spectrometry can be performed in order to answer additional questions like whether FPD mutants phosphorylate the same residue of Rab10. After its development, this technique paved the way for researchers in the field of Parkinson research to explore the endogenous levels of Rab10 phosphorylation in cultured cells as well as in tissues. Don't forget that working with microcystin added to the lysis buffer can be extremely hazardous and precautions such as wearing appropriate personal protective equipment should always be taken while preparing cell lysates.
