ELISA Cell-Based Dose Response to Identify Inhibitors of Alpha-Synuclein Translation in SH-SY5Y Cells
Jack T. Rogers, Massachusetts General Hospital, firstname.lastname@example.org, 617-726-8838, Charlestown, MA ..more
BioActive Compound: 1
Depositor Specified Assays
Broad Institute MLPCN Alpha-Synuclein 5'UTR Project
Project ID: 2023
Keywords: alpha-synuclein, Parkinson's disease, translation, RNA stem-loop, 5'-untranslated region
Primary Collaborators (and laboratory where this assay was performed):
Jack T. Rogers, Massachusetts General Hospital, email@example.com, 617-726-8838, Charlestown, MA
The goal of this project is to identify novel small molecule probes that inhibit alpha-synuclein translational expression in dopaminergic neurons by targeting the 5'-untranslated region (5'UTR) stem-loop of alpha-synuclein as a major new therapeutic target to retard the progression of Parkinson's disease (PD). The 5'UTR of alpha-synuclein mRNA can interact with Iron Regulatory Protein-1 (IRP1), which upon interaction causes an increase in alpha-synuclein mRNA translation. Probes that can successfully reduce alpha-synuclein expression levels as measured in this luciferase reporter assay will be further tested for specificity in cells lacking the alpha-synuclein 5'UTR stem-loop to confirm that probes are acting through the intended target. Probe selectivity will be tested in cells containing the prion protein 5'UTR mRNA stem-loop, which is fused to a luciferase reporter.
In the ELISA orthogonal screen confirmation of alpha-synuclein tranlation inhibitors was sought. The ELISA used SH-SY5Y neuroglioblastoma cells that were not transfected. Compound was tested in 4-point dose in an effort to observe a dose-response effect.
Inhibitors of alpha-synuclein translation should decrease alpha-synuclein protein levels.
ELISA Protocol: (Invitrogen alpha-synuclein ELISA kit #KHB0061)
- Plate 6000 cells/well (50 uL) with phenol red free media
- Incubate 24 hours at 37C, 5% CO2, 95% humidity
- Pin 100 nL of compounds
- Incubate 48 hours at 37C, 5% CO2, 95% humidity
- Add 5uL Alamar blue solution per well, Incubate for 1 Hr at 370C, 5% CO2, 95% humidity
- Read for fluorescence (570nm excitation, 585nm emission)
- Remove media
- Add cell lysis solution and keep the plates on plate shaker for 1 Hr
- Remove 20 uL lysate and place in 500 uL epi tube
- Centrifuge the cell lysate at 13,000 rpm for 10 min
- Transfer 12 uL supernatant to 48 uL of standard diluent buffer (1:5)
- Add 50 uL of Standard curve and other samples on ELISA plate
- Wait 5 min.
- Pipette 50 uL of anti-alpha-Synuclein (Detection Antibody) solution, tap plate to mix
- Cover wells with plate cover and incubate for 3 hours at room temperature
- Wash wells 4 times with wash buffer
- Add 100 uL anti-rabbit IgG-HRP antibody solution
- Cover wells with the plate cover and incubate for 30 mins at RT
- Wash wells 4 times
- Add 100 uL of Stabilized Chromogen
- Keep sealed for 30 minutes at room temperature in the dark
- Add 100 uL of Stop Solution, tap plate to mix
- Read the absorbance of each well at 450 nm
Dose Response Data Analysis:
Three independent wells were evaluated for each compound concentration. Negative control condition (DMSO) was included.
Live Cell Correction Factor at dose was calculated as:
OD Alamar Blue at dose/ OD Alamar Blue at DMSO
ELISA values are the average of the independent wells for each compound concentration. The ELISA included a standard curve of recombinant alpha-synuclein (provided in the kit). The ELISA value is background normalized to the ELISA kit using the zero condition as described in the kit manual. ELISA values for each compound concentration are adjusted for live cells as follows:
OD ELISA at dose/ Live Cell Correction Factor at dose
The alpha-synuclein concentration values are extrapolated from the recombinant alpha-synuclein standard curve fit equation. Percent inhibition of alpha-synuclein was calculated based on the assumption that complete (100%) inhibition is attainable, as follows:
100 - (100 * (alpha-synuclein concentration at dose/ alpha-synuclein concentration at DMSO))
EC50 was determined to be the lowest dose that supported >= 50% of Maximum Observed Activity.
Inactive compounds = 0
Active compounds = -10 * Log(EC50)
Activity_Outcome = 1 (inactive)
Dose Response is not observed.
Activity_Outcome = 2 (active)
Dose Response is observed with an EC50 < 0.2uM.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)