Feb 10, 2025
- 1Stanford University
Protocol Citation: Ching-Chieh Chou, Judith Frydman 2025. Aβ ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9e6plk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 10, 2025
Last Modified: February 10, 2025
Protocol Integer ID: 119915
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Aligning Science Across Parkinson's
Grant ID: ASAP-024268
Abstract
Measure Aβ42 levels in human neurons.
Human Aβ42 ELISA Kit (Thermo Fisher Scientific).
Human tNeurons are first trypsinized, washed with ice-cold PBS
and pelleted by centrifugation for 5 min at 1,000 X G.
Cells are lysed with RIPA buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate) supplemented with protease inhibitors (Roche).
Collect the supernatants and protein concentrations are determined by BCA assay (Thermo Fisher Scientific).
Human Aβ42 ELISA Kit is used to detect and quantify the Aβ42 levels in total tNeuron lysates.
Prepare standards of 1000, 500, 250, 125, 62.5, 31.25, 15.63, and 0 pg/mL human Aβ42 in Standard Diluent Buffer.
50 µL of standards, control medium, cell lysates are added to each well of a 96-well plate.
Add 50 µL anti-human Aβ42 antibody per well and incubate 3 hr in the dark at room temperature with shaking.
Remove the solution and rinse wells 4 times with 1X Wash Buffer.
Add 100 µL Anti-Rabbit IgG HRP into each well and incubate in the dark for 30 min at room temperature.
Remove the solution and rinse wells 4 times with 1X Wash Buffer.
Add 100 µL Stabilized Chromogen to each well and incubate in the dark for 30 min at room temperature.
Add 100 µL Stop Solution to each well. The plate is analyzed within 2 hr according to manufacturer’s protocol and Aβ42 values are normalized to total protein concentration of lysates.