Aug 26, 2023
Membrane and cytosol fractionation
- 1Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
Protocol Citation: wusj, schekman 2023. Membrane and cytosol fractionation. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm3d7l5b/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: June 26, 2023
Last Modified: August 26, 2023
Protocol Integer ID: 84011
Funders Acknowledgement:
Randy Schekman
Grant ID: Howard Hughes Medical Institute
Abstract
This protocol describes membrane and cytosol fractionation of cells expressing different DNAJC5 isoforms
Cytosol fractionation
Cytosol fractionation
Cells (one 10 cm dish) were cultured to 70% confluence and transfected with different constructs
of DNAJC5
One day after transfection, we harvested the transfected cells by scraping in 1 ml B88 (20 mM HEPES-KOH, pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, and 5 mM magnesium acetate) plus a cocktail of protease inhibitors
Cells were homogenized by 10 passages through a 22G needle
Homogenates were centrifuged at 500×g for 00:10:00 and the resulting post-nuclear supernatant (PNS) fractions were centrifuged at 100,000×g for 01:30:00
1h 40m
High-speed supernatant fractions were then subjected to a repeat centrifugation to achieve a clarified cytosol fraction
The pellet fraction was washed and resuspended in the same volume of B88
Resuspended material was also centrifuged again to collect a washed membrane fraction
Membranes were lysed in lysis buffer
Membrane fractionation
Membrane fractionation
The PNS was subjected to differential centrifugation at 3000×g for 00:10:00
10m
The supernatant was centrifuged at 25,000×g for 00:20:00
20m
The supernatant was centrifuged at 100,000×g for 00:30:00
30m
Membrane fractions were normalized to phosphatidylcholine content and analyzed by immunoblot
Proteinase K protection assays
Proteinase K protection assays
25m
The 25,000×g membrane fraction was aliquoted into three tubes: one without proteinase K, one with proteinase K (10 μg/ml), and one with proteinase K plus TritonX-100 (0.5%)
The incubation was conducted On ice for 00:20:00
20m
The reaction was stopped by sequential addition of PMSF (1 mM)
Add sample buffer. Then, samples were then heated on metal block at 95 °C for 00:05:00 and analyzed by SDS-PAGE and immunoblot.
5m