Jun 28, 2024

Public workspaceCrude Subcellular fractionation of FAM177A1-GFP expressing cells

DOI
dx.doi.org/10.17504/protocols.io.n2bvjn2ypgk5/v1
  • 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
Berrak Ugur
Open access
DOI: dx.doi.org/10.17504/protocols.io.n2bvjn2ypgk5/v1
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
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol details the crude subcellular fractionation of FAM177A1-GFP expressing cells.
Materials
Fractionation buffer:
AB
Tris, pH 7.425 mM
NaCl150 mM
Protease inhibitor
Crude Subcellular fractionation
Crude Subcellular fractionation
2d 1h 5m

Note
All preparations were performed TemperatureOn ice .

Culture and transfect HeLa cells as described in dx.doi.org/10.17504/protocols.io.eq2lyp55mlx9/v1

Duration24:00:00 -Duration48:00:00 after transfection, wash cells in 6 well plates with PBS.

2d
Wash
Add Amount200 µL PBS (or fractionation buffer) to each well.

Pipetting
Scrape cells to release from well and transfer to a 1.7 mL eppendorph tube with additional Amount100 µL fractionation buffer wash.

Spin the lysate at Centrifigation1500 rpm, 00:05:00 in a benchtop centrifuge.

5m
Centrifigation
Remove the supernatant and resuspend cell pellet in Amount1 mL of cold fractionation buffer.

Homogenize resuspended cells with cell cracker (Isobiotec; 8-12 strokes), Amount1 mL at a time.

Wash with Amount1 mL fractionation buffer between samples.

Wash
Always use new syringes for each sample.
Ultracentrifuge lysates in a Beckman-Coulter table-top ultracentrifuge (TLA100 rotor) at Centrifigation50000 rpm, 4°C, 01:00:00 to pellet membrane.

1h
Centrifigation
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.