Jun 28, 2024
Crude Subcellular fractionation of FAM177A1-GFP expressing cells
- Berrak Ugur1,2,3,4,
- Michael G. Hanna1,2,3,4,
- Pietro De Camilli1,2,3,4
- 1Departments of Cell Biology and Neuroscience, Yale University School of Medicine, New Haven, CT, USA;
- 2Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT, USA;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
- 4HHMI, Yale University School of Medicine, New Haven, CT, USA
Protocol Citation: Berrak Ugur, Michael G. Hanna, Pietro De Camilli 2024. Crude Subcellular fractionation of FAM177A1-GFP expressing cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn2ypgk5/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, 2024
Last Modified: June 28, 2024
Protocol Integer ID: 102587
Keywords: ASAPCRN, crude subcellular fractionation of fam177a1, crude subcellular fractionation, fam177a1, expressing cell, cells this protocol detail, cell, gfp
Funders Acknowledgements:
ASAP
Grant ID: 000580
Abstract
This protocol details the crude subcellular fractionation of FAM177A1-GFP expressing cells.
Materials
Fractionation buffer:
| A | B | |
| Tris, pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| Protease inhibitor | ||
Crude Subcellular fractionation
2d 1h 5m
Note
All preparations were performed On ice .
24:00:00 -48:00:00 after transfection, wash cells in 6 well plates with PBS.
2d
Add 200 µL PBS (or fractionation buffer) to each well.
Scrape cells to release from well and transfer to a 1.7 mL eppendorph tube with additional 100 µL fractionation buffer wash.
Spin the lysate at 1500 rpm, 00:05:00 in a benchtop centrifuge.
5m
Remove the supernatant and resuspend cell pellet in 1 mL of cold fractionation buffer.
Homogenize resuspended cells with cell cracker (Isobiotec; 8-12 strokes), 1 mL at a time.
Wash with 1 mL fractionation buffer between samples.
Always use new syringes for each sample.
Ultracentrifuge lysates in a Beckman-Coulter table-top ultracentrifuge (TLA100 rotor) at 50000 rpm, 4°C, 01:00:00 to pellet membrane.
1h
After the centrifugation, transfer the supernatant to a new 1.7 mL Eppendorf tube and save it for western blot analysis.
Solubilize the membrane fractions from the bottom of the tubes using 4X Laemni buffer for western blot analysis.