The overall goal of this methodology is to extract soluble and insoluble alpha-synuclein from post-mortem Parkinsonian brains. This method can help answer key questions in the study of Parkinson's disease. The main advantage of this technique is that it is a relatively straightforward technique using common laboratory reagents and instruments.
After preparing the necessary solutions and buffers, begin by placing a 0.5 gram sample of frozen basal ganglia tissue onto a small Petri dish. Use a sterile scalpel blade to rapidly mince into one millimeter square fragments. Collect the minced tissue into a 15 milliliter tube, and add 10 volumes of ice cold, 1X TBS to the tube.
Homogenize the samples using a mechanical homogenizer at 20, 000 rpm for 10 seconds. Then cool on ice for two minutes. Repeat the homogenization three times, cooling on ice each time.
Centrifuge the homogenate at 10, 000 gs for five minutes at four degrees Celsius. Transfer the crude homogenate supernatant to polycarbonate centrifuge tubes, and centrifuge at 100, 000 gs for one hour at four degrees Celsius. Transfer the supernatant to cooled tubes on ice, and retain this as the TBS soluble fraction.
Wash in five volumes of 1X TBS buffer, and centrifuge at 100, 000 gs for 15 minutes at four degrees Celsius. Aspirate and discard the supernatant. Then repeat the wash.
Thorough washing of pellets in between ultracentrifugation steps is critical for the success of this procedure. After allowing the samples to reach a temperature of 10 degrees Celsius to avoid SDS precipitation, add five volumes of 1X TBS-SDS and re-suspend the final pellet by sonicating for 10 seconds at 20 kilohertz at room temperature. Ultracentrifuge at 100, 000 gs for 30 minutes at 25 degrees Celsius.
After ultracentrifugation, transfer the supernatant to cooled tubes on ice, and retain as the SDS soluble fraction. Add five volumes of room temperature 1X TBS-SDS buffer to the pellet, and centrifuge at 100, 000 gs for 15 minutes at 25 degrees Celsius. Discard the wash buffer, and repeat the wash.
After discarding the supernatant, add 50 microliters of 1X TBS-SDS-Urea and solubilize using a sonicator set at 20 kilohertz for 10 seconds. Label this sample as the urea soluble fraction. It is essential to thoroughly ensure all the insoluble proteins are extracted in the procedure.
Dilute TBS-SDS-Urea samples one-to-one with 1X TBS buffer to allow compatibility with protein assay reagents, and assay each fraction for total protein content using a commercial protein assay kit according to the manufacturer's protocol. Aliquot samples into 20 microliter aliquots with 10%glycerol to reduce future freeze-thaw cycles, and store at negative 80 degrees Celsius. Load 10 microliters of the TBS, TBS-SDS, and TBS-SDS-Urea extracts in a molecular weight marker on 10 well, four to 12 percent Bis-Tris polyacrylamide gel with MOPS as running buffer using standard techniques.
Run the gel at 200 volts for one hour, or until the blue dye from loading buffer has reached the bottom of the gel. Wet a nylon membrane in 100%methanol, and then soak in transfer buffer containing 20%methanol. Make an identifying mark on the blot to determine the protein side and the orientation of the molecular weight size markers.
Sandwich the gel with the membrane alongside wet filter paper. Perform the transfer for two hours at 40 volts. Block the in 5%BSA solution in 1X PBS-T for 30 minutes with shaking at 70 rpm to prevent non-specific binding of primary antibody to the membrane.
Probe the protein blot by incubating with six milliliters of the alpha-synuclein primary antibody Syn-1 diluted one to 750 in PBS-T overnight at four degrees Celsius. The next day, wash the membrane with 1X PBS-T three times for five minutes each time. Then incubate the blot with the appropriate HRP conjugated secondary antibody diluted one to 2, 000 in PBS-T for 30 minutes.
Wash the membrane as before with 1X PBS-T. Immerse the blots in enhanced chemiluminescence solution according to the manufacturer's instructions in a dark room. Cover the blots with cling film, and capture the signal using autoradiography.
After imaging, incubate the western blot with six milliliters of western blot stripping buffer for 10 minutes with constant shaking to strip the blot of alpha-synuclein antibody signal. Repeat the western blot procedure to probe the membrane with beta-actin primary antibody at a one to 8, 000 dilution. Scan and convert the final image into a Tiff image and measure densities using NIH ImageJ for quantitation purposes.
The following images are representative immunoblots of alpha-synuclein levels from PD and control human basal ganglia tissue. The TBS, SDS, and urea soluble fractions were analyzed for the presence of alpha-synuclein using western immunoblot. 10 milligrams of protein was loaded into each lane.
In the TBS fraction, alpha-synuclein monomers are present in all cases. The asterisk possibly represents a C-terminally truncated form of alpha-synuclein. In the SDS fraction, some oligomers of alpha-synuclein are seen along with monomers, indicated by the solid arrowhead in the PD tissue.
In the urea fraction, monomers, oligomers, and aggregated alpha-synuclein are variably expressed in the PD cases, reflecting their respective Lewy body load. The neurologically normal control samples show the presence of only small amounts of monomeric alpha-synuclein. Once mastered, this protein extraction technique can be done within a working day, followed by western immunoblot the next day, if it is performed properly.
After its development, this technique paved the way for researchers in the field of Lewy body biology to explore insoluble and aggregated alpha-synuclein species in pathological human tissue and in in-vivo animal models. Don't forget that working with unfixed human tissue requires precautionary measures, and gloves and laboratory coats should be worn throughout for all the handling procedures.
