Mar 04, 2022
TSS, TXS, SDS Serial Extraction
- 1University of Ottawa
Protocol Citation: Haley Geertsma 2022. TSS, TXS, SDS Serial Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.b5vvq666
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: March 03, 2022
Last Modified: March 04, 2022
Protocol Integer ID: 59029
Abstract
This protocol is used to serially extract protein from mouse brain tissue in increasingly stringent buffers.
Weigh brain sample and add 3X volume of TSS buffer + 1X protease inhibitor.
TSS Buffer: 140mM NaCl + 5mM Tris-HCl in H2O
2m
Homogenize in a dounce homogenizer then transfer to a clean tube.
2m
Centrifuge at 21130g for 30 minutes at 4oC then save the supernatant as 'TSS fraction'.
30m
Resuspend the pellet in the same volume of TSS buffer then centrifuge at 21130g for 30 minutes at 4oC and discard the supernatant.
35m
Resuspend the pellet in the same volume of TXS buffer then incubate on ice for 10 minutes.
TXS Buffer: TSS buffer + 0.5% Triton X-100
15m
Centrifuge at 21130g for 30 minutes at 4oC then save the supernatant as 'TXS fraction'.
30m
Resuspend the pellet in the same volume of TSS buffer then centrifuge at 21130g for 30 minutes at 4oC and discard the supernatant.
35m
Resuspend the pellet in the same volume of SDS buffer then incubate at room temperature for 10 minutes.
SDS Buffer: TSS buffer + 1% sodium dodecyl sulfate (SDS)
15m
Centrifuge at 21130g for 30 minutes at room temperature then save the supernatant as 'SDS fraction'.
30m