This article details a protocol for quantifying mutated ��-synuclein in brain tissue from transgenic mice. The method involves homogenization, centrifugation, and enzyme-linked immunosorbent assay (ELISA) techniques.
Begin with the brain tissue from a transgenic mouse overexpressing mutated α-synuclein, a misfolded neuronal protein associated with neurodegenerative disease, immersed in a high-salt buffer.
Homogenize the tissue with a large pestle, followed by a small pestle to disrupt the tissue and release intracellular proteins, including α-synuclein.
Transfer the homogenate to a tube and centrifuge. Collect the protein-containing supernatant and dilute it with a buffer.
Add this supernatant to an α-synuclein-specific antibodies-coated microplate pretreated with a blocking agent to prevent nonspecific binding.
Incubate with agitation, allowing normal and mutated α-synuclein to bind.
Wash the plate, then add detection antibodies specific to mutated α-synuclein and incubate with agitation.
Wash again and add enzyme-linked antibodies, followed by incubation under agitation.
Add substrate and incubate in the dark under agitation. The substrate reacts with the enzyme to produce a color change.
Stop the reaction with an acid and measure the absorbance to quantify mutated α-synuclein.
To prepare brain homogenates, add an adequate volume of HS buffer to the dissected brain regions to reach an expected percent homogenate, as shown in the table below. Using a tissue grinder composed of borosilicate glass tube and two pestles, A and B, pour a brain region to be crushed into the tube. And then with pestle A, homogenize the tissue 10 times to dissociate it.
Then, switch to pestle B and homogenize an additional 20 times. With a transfer pipette, transfer the homogenate into a 1.5-milliliters tube, repeating the homogenization with the remaining brain samples. Centrifuge the samples at 1,000 g for five minutes, at four degrees Celsius. And then collect the supernatants and divide them into 200-microliter aliquots.
With 100 microliters of the antibody coating solution, coat the wells of 96 well microplates and incubate at four degrees Celsius overnight. The following day, add 300 microliters of PBST to the wells, and use a plate washer to wash the plates five times. While working at room temperature, moving forward, add 200 microliters of T20 PBS blocking buffer to each well, and shake the plates at 150 RPM for one hour before using PBST to wash the plates five times.
Using PBST, 1% of BSA, dilute the brain homogenates using the guidelines listed here. And add 100 microliters to each well. Then incubate at room temperature and 150 RPM for two hours before using PBST to wash the plates five times.
After the final wash, add the necessary alpha synuclein detection antibodies diluted in PBST and 1% BSA as listed in table two of the text protocol. Incubate at room temperature and 150 RPM for one hour before washing the plates five times as before.
Next, add either anti-mouse or anti-rabbit IGG HRP conjugates diluted to 1 to 8,000 in PBST, 1% BSA. And incubate at 150 RPM for one hour before washing the plates five times. Then add 100 microliters of TMB solution to each well, and incubate at 150 RPM for 15 minutes in the dark. Stop the reaction by adding 100 microliters of one normal hydrochloric acid per well. Then, use a microplate reader to measure the absorbance at 450 nanometers.
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