Jul 09, 2024

Public workspaceMicrosomal membrane isolation

 Forked from Microsomal membrane isolation from cell culture
DOI
dx.doi.org/10.17504/protocols.io.5qpvo3w7dv4o/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson’s (ASAP)
Rania Abou El Asrar
Open access
DOI: dx.doi.org/10.17504/protocols.io.5qpvo3w7dv4o/v1
Protocol Citation: Rania Abou El Asrar, Peter Vangheluwe 2024. Microsomal membrane isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3w7dv4o/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
This protocol is currently being used to assess the activity of P5B-ATPases.
Created: December 06, 2023
Last Modified: July 09, 2024
Protocol Integer ID: 91888
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Abstract
Microsomal membrane isolation
Harvest cells
Harvest cells
Harvest cells by detachment with phosphate buffered saline (PBS, Sigma, D8537-500ml) + 0.2% EDTA.

Pellet cells by centrifugation (5 min, 400 gavg, 4°C).
Wash cell pellet in phosphate buffered saline (PBS, Sigma, D8537-500ml). Repeat 2 times.
Pellet cells by centrifugation (5 min, 400 gavg, 4°C).
Lyse cells
Lyse cells
Resuspend pellet in hypnotic LIS buffer (10 mM Tris HCl pH 7.5, 0.5 mM MgCl2, 1 mM DTT) supplemented with protease inhibitor (Sigmafast, Sigma, S8830-20TAB).
Incubate 15 min on ice.
Transfer the suspension to a dounce homogenizer and complete 60 up-and-down strokes.
Add an equal volume of 1 M buffer (0.5 M sucrose, 10 mM Tris HCl, pH 7.5, 40 µM CaCl2, 1 mM DTT) supplemented with protease inhibitor.
Complete another 30 up-and-down strokes.
Centrifuge lysate (10min, 1000 gavg, 4°C) to pellet and remove nuclear fraction.
Collect supernatant to continue isolation.
Isolate microsomal fraction
Isolate microsomal fraction
Centrifuge supernatant from previous step (20min, 15 000 gavg, 4°C) to pellet the mitochondrial/lysosomal fraction.
Collect supernatant to continue isolation (pelleted mitochondrial/lysosomal fraction can be stored for future analysis).
Centrifuge supernatant from previous step (35 min, 140 000 gavg, 4°C) to pellet the microsomal fraction.
Resuspend pellets in 0.25 M sucrose, 1 mM DTT and supplemented with protease inhibitors.
Snap freeze the resuspended mitochondrial/lysosomal and microsomal fractions in liquid nitrogen.
Store at -80 °C.