TSS, TXS, SDS Serial Extraction
Haley Geertsma
Abstract
This protocol is used to serially extract protein from mouse brain tissue in increasingly stringent buffers.
Steps
Weigh brain sample and add 3X volume of TSS buffer + 1X protease inhibitor.
TSS Buffer: 140mM NaCl + 5mM Tris-HCl in H2O
Homogenize in a dounce homogenizer then transfer to a clean tube.
Centrifuge at 21130g for 30 minutes at 4oC then save the supernatant as 'TSS fraction'.
Resuspend the pellet in the same volume of TSS buffer then centrifuge at 21130g for 30 minutes at 4oC and discard the supernatant.
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
Centrifuge at 21130g for 30 minutes at 4oC then save the supernatant as 'TXS fraction'.
Resuspend the pellet in the same volume of TSS buffer then centrifuge at 21130g for 30 minutes at 4oC and discard the supernatant.
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)
Centrifuge at 21130g for 30 minutes at room temperature then save the supernatant as 'SDS fraction'.
