Oct 02, 2023
Biochemical assays and evaluation
- lparra1
- 1University of California San Diego
Protocol Citation: lparra 2023. Biochemical assays and evaluation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pey7g2w/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: October 02, 2023
Last Modified: October 02, 2023
Protocol Integer ID: 88652
Abstract
Preparation of brain and Neuro2A lysates
Preparation of Brain and Neuro2A Lysates
Preparation of Brain and Neuro2A Lysates
Whole mouse brains were homogenized with a Dounce tissue grinder (20
times) in neuronal protein extraction reagent (N-PER) (Thermo Scientific)
containing protease/phosphatase inhibitors (Cell Signaling #5872).
Triton X-100 (Sigma #X100-100ML) was added at
a final concentration of 1%, and the samples were incubated with rotation for 1
hour at 4 °C.
Samples were centrifuged at 10,000 × g
for 10 min at 4 °C, and the supernatant was collected.
For Neuro2A lysates, cells were washed with 1X PBS 3 times and incubated
for 5 min on ice in the presence of N-PER reagent supplemented with protease
inhibitors.
Samples were centrifuged at 10,000 × g
for 10 min at 4 °C to remove cellular debris.
Total protein
concentration was measured in brain and Neuro2A lysates (DC‱ Protein Assay Kit II, Biorad), and samples were
used in subsequent experiments.