The overall goal of this fractionation protocol is to enrich detergent-insoluble protein aggregates from human postmortem brain tissue. So this method can help answer key questions in the field of neurodegeneration, such as the identification and characterization of misfolded protein aggregates in disease. The main advantage of this technique is the fascial enrichment and isolation of detergent-insoluble protein aggregates from brain tissue within a short time frame.
Demonstrating this procedure will be Tram Nguyen, a technician from my laboratory. Obtain frozen postmortem brain tissue from healthy control and pathologically confirmed AD.Use forceps and a razor blade to excise approximately 250-milligram portions of gray matter from each tissue sample. Allow the brain segment to thaw for one minute.
Now, dice a piece of tissue into roughly two-millimeter cubed pieces as it thaws. Transfer to a two-milliliter prechilled Dounce homogenizer tube on ice. Remember to avoid white matter and any large blood vessels.
Work quickly and place the tissue on TARE disposable weigh boats. Take care to prevent the tissue from thawing by frequently placing the weighing boat with brain tissue into polystyrene containers with dry ice. After dissecting and weighing all required pieces of tissue, remove a weigh boat containing a piece of tissue from dry ice.
Next, add five milliliters of ice cold low-salt buffer with protease and phosphatase inhibitor cocktail per gram of tissue, and homogenize on ice using approximately 10 strokes of high-clearance pestle A and 15 strokes of low-clearance pestle B.Then use a nine-inch glass Pasteur pipette to transfer each homogenate to a labeled two-milliliter polypropylene tube. Aliquot the remaining homogenate into additional labeled 1.5-milliliter tubes in a similar fashion for storage at minus 80 degrees Celsius. To proceed with fractionation, add 100 microliters of five molar sodium chloride and 100 microliters of 10 percent sarkosyl to each 0.8-milliliter aliquot.
Mix well by inversion. And then incubate on ice for 15 minutes. Following the incubation, use a sonicator equipped with a microtip probe to deliver three five-second pulses at 30 percent amplitude.
After using the bicinchoninic acid method to determine the protein concentrations of the homogenates, add ice cold sark buffer with protease and phosphatase inhibitors to each sample to a final concentration of 10 milligrams per milliliter. Then transfer 500 microliters of each sample into 500-microliter polycarbonate ultracentrifuge tubes. Load tubes into a prechilled rotor and ultracentrifuge at 180, 000 times g for 30 minutes at four degrees Celsius.
Following the centrifugation, transfer the S1 sarkosyl-soluble supernatants to 1.5-milliliter tubes and store at minus 80 degrees Celsius. Add 200 microliters of sark buffer to the ultracentrifuge tubes containing the detergent-insoluble P1 fractions and use the pipette tip to dislodge the pellets from the bottom of the ultracentrifuge tubes. Next, transfer the re-suspended P1 pellets to new 500-microliter ultracentrifuge tubes, pair-balance, and centrifuge at 180, 000 times g for an additional 30 minutes at four degrees Celsius.
Discard the S2 supernatant. Then add 50 to 75 microliters of urea buffer with protease and phosphatase inhibitor cocktail to the sarkosyl-insoluble pellets and incubate for 30 minutes at room temperature to solubilize the P2 pellet. After the incubation, transfer the re-suspended P2 pellets to labeled 0.5-milliliter tubes and fully solubilize the pellets, using a brief one-second microtip sonication at 20 percent amplitude.
Determine the protein concentrations of the sarkosyl-soluble and sarkosyl-insoluble fractions using the BCA assay method. Prepare 40-microgram samples of total homogenate sark-soluble S1 and sark-insoluble P2 fractions in one-x Laemmli SDS-PAGE sample buffer with five millimolar TCEP at pH seven. Incubate samples at 95 degrees Celsius for five minutes.
Load the samples onto precast 10-well four to 12 percent Bis-Tris SDS-PAGE gels. Electrophorese at 80 volts for the first centimeter and 120 volts for about an hour or until the tracking dye reaches the bottom of the gel. Submerge the precast gel cassettes into distilled water and then use a fresh razor blade and gel knife to split open.
Gently cut away the combs and very bottom of each gel with a fresh razor blade. Transfer to 0.2 micron nitrocellulose membranes using a dry transfer method on a blotting machine. After transfer, block the membranes in blocking buffer without Tween-20 for 30 minutes, followed with blocking buffer with 0.5 percent Tween-20 for 30 minutes.
After blocking, rinse the membranes in TBS with 0.1 percent Tween-20 for five minutes to remove excess blocking buffer. Prepare 1:1, 000 dilutions of the primary antibodies in blocking buffer with 0.5 percent Tween-20. Phospho-Tau pT231 and EEA1 are used here.
Incubate the membranes in primary antibody solution overnight at four degrees Celsius with circular agitation. The next day, rinse the membranes in TBS with 0.1 percent Tween-20 three times for 15 minutes each. Probe the membranes with a 1:20, 000 dilution of the fluorescently-labeled near-infrared secondary antibody and blocking buffer with 0.5 percent Tween-20 for 60 minutes at room temperature in darkness on a shaker.
Following the incubation, rinse the membrane three times for 10 minutes in TBS with 0.1 percent Tween-20 and twice in TBS for five minutes each time. Scan the membrane using an infrared imaging system at the appropriate excitation wavelength for the secondary antibody used. This Coomassie-stained gel shows 20 mcg of protein from TH-S, S1, S2, and P2 fractions.
Western blot Analysis clearly demonstrates that in AD brain, the pathologic high-molecular weight phosphorylated Tau aggregates with sarkosyl-insoluble and very little phospho-Tau remained in the sarkosyl fractions. EEA1 was used as a general marker for most native nonpathologic proteins that are inherently sarkosyl-soluble and partitioned into the S1 fraction in both control and AD brain. Notably, there was slightly less signal for EEA1 in the soluble fraction compared to the TH-S fraction, indicating that a minor pool of EEA1 is likely sarkosyl-insoluble, yet below the limit of detection by western blot with this particular antibody.
This figure shows the proteomic analysis of pathologic AD proteins using a single-step or multi-step fractionation protocol. The single-step fractionation technique may be advantageous for low-abundance proteins, as the overall sample losses are significantly reduced. While attempting this procedure, it's important to remember to excise as much white matter from the brain tissue as possible.
Following this procedure, other methods like mass-spectrometry-based proteomics can be performed in order to identify and characterize core protein components of pathological aggregates in their degenerative diseases.
