Bitan:Novex ELISA Human alpha-Synuclein

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1. Purchase Novex Human alpha-Synuclein kits (catalog number: KHB0061) through ThermoFisher Scientific. Receive on dry-ice and store at 4°C. Follow kit-provided instructions to perform the analyses. View the following video for general understanding of procedure Note, follow kit-provided incubation temperatures and times.

2. Prior to analysis, pull samples from the -20°C freezer and thaw over ice. Allow all solutions to come to room temperature prior to use, but only pull from fridge within one hour of use.

3. Reconstitute α-synuclein Standard in Standard Diluent Buffer at 15 ng/mL by 1.30mL of Standard Diluent Buffer to α-synuclein Standard. Swirl gently, and let sit for ten minutes to ensure complete reconstitution.

4. Prepare serial dilutions in clean microcentrifuge tubes. Prepare additional standards at concentrations of 7.5, 3.75, 1.88, 0.94, 0.47 and 0.23 ng/mL. 0ng/mL is an empty tube. Label the first microcentrifuge tube "15ng/mL" and add 500uL of standard. Prepare 1:1 serial dilutions, using Standard Diluent Buffer to prepare remaining standards. Note, standard curve is run in duplicate, and a new standard curve is required for each analysis.

5. Next, prepare samples. 1:1000 dilution is appropriate for Buffer-Soluble fractions, and 1:500 for Buffer-Insoluble fractions. Prepare all sample dilutions using Standard Diluent Buffer. Ensure there is enough volume to run samples in duplicate (50uL of sample per well). All standards and samples are run in duplicate.

6. Add 50μL of Standard Diluent Buffer to each well, excluding the two wells reserved for the 0ng/mL standard (Chromagen blank). These Chromagen Blank wells will be left empty.

7. Next, add 50μL of Standards or pre-diluted samples to each micrometer well, leaving 0ng/mL (Chromagen Blank) standard empty.

8. Add 50μL of Human α-synuclein Detection Antibody too each well, except the 0 ng/mL Chromagen blank. Cover plate with an adhesive strip, tap gently on sides to mix, and incubate at room temperature (approximately 25°C) for three hours. During the incubation, prepare 1L of Wash Buffer by diluting Wash Buffer Concentrate, (25X) 1:25 in Milli-Q water, and store in a clean wash-bottle. Additionally, prepare 12mL of Anti-Rabbit IgGHRP Working Solution by diluting Anti-Rabbit IgGHRP, (100X) solution 1:100 in HRP Diluent.

9. After 3-hour incubation, remove adhesive strip and perform the Wash Step four times. To do so, invert the plate, dump contents in waste container, and tap dry on a paper towel. Using a squirt bottle, flood each well with Wash Buffer Solution and let sit for 10 seconds. Invert and tap dry on a paper towel and repeat the wash step three additional times.

10. After, pipette 100μL of Anti-Rabbit IgGHRP Working Solution to each well, except the two Chromagen Blank wells. Cover with an adhesive strip and incubate at room temperature for thirty minutes.

11. After incubation, perform wash step four times, and tap dry on a paper towel.

12. Add 100μL of Stabilized Chromagen to each well, including the Chromagen Blanks. Incubate at room temperature for thirty minutes in the dark, without an adhesive strip. Cover with cardboard box. Be sure not to cover with aluminum foil.

13. After incubation, add 100μL of Stop Solution to each well and tap gently to mix.

14. Use a microplane reader to read absorbances at 450nm. Read absorbances within two-hours of adding stop solution.

15. Plot standards and absorbances to determine linear equation and concentration of unknown samples, being sure to account to dilution factors of samples.