Aug 12, 2022

Public workspaceCrude Membrane Fractionation of Cultured Cells

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmnb99g3p/v1
  • Asad Malik1,
  • Dario R Alessi1,
  • Suzanne Pfeffer2
  • 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.;
  • 2Stanford University School of Medicine, Stanford, CA 94305-5307
  • asap
Dario R Alessi
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmnb99g3p/v1
External link: https://doi.org/10.1042/BCJ20220308
Protocol Citation: Asad Malik, Dario R Alessi, Suzanne Pfeffer 2022. Crude Membrane Fractionation of Cultured Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmnb99g3p/v1
Manuscript citation:
Malik AU, Karapetsas A, Nirujogi RS, Chatterjee D, Phung TK, Wightman M, Gourlay R, Morrice N, Mathea S, Knapp S, Alessi DR, PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the COR GTPase domain. Biochemical Journal 479(18). doi: 10.1042/BCJ20220308
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 16, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 64712
Keywords: Cultured Cells, Crude Membrane Fractionation, Immunoblotting analysis, ASAPCRN, crude membrane fractionation of cultured cell, insoluble fraction from cultured cell, crude membrane fractionation, cultured cell, crude cellular extract, membrane versus cytosolic distribution, signaling pathway, protocol for fractionating, cytosol, cell, components from lrrk1, containing insoluble fraction, membrane, enriched fraction, cytosolic distribution, fractionating
Abstract

We present here a protocol for fractionating crude cellular extracts to prepare membrane and cytosol-enriched fractions and a nuclei-containing insoluble fraction from cultured cells. We deploy this protocol for determining the membrane versus cytosolic distribution of components from LRRK1 and LRRK2 signaling pathways.
Note
We recommend analysing the products of this fractionation scheme by quantitative immunoblotting (as described indx.doi.org/10.17504/protocols.io.6qpvr68e3vmk/v1).

Note
This protocol was adapted from https://doi.org/10.15252/embj.201798099

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Materials
MATERIALS

Reagents:

Buffer A:

Concentration10 millimolar (mM) HEPES Ph7.4 and cOmpleteTM EDTA-free Protease Inhibitor Cocktail
Note
Added fresh before use,ReagentcOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)



Buffer B:

AB
HEPES pH 7.4250 mM
Sodium Chloride750 mM
Magnesium Chloride25 mM
DTT2.5 mM
GDP500 nM
Sodium Fluoride250 mM
Sodium Pyrophosphate25 mM
Microcystin-LR (Enzo Life Sciences, ALX-350-012)5 μg/ml
cOmpleteTMEDTA-free Protease Inhibitor Cocktail (added fresh before use, Roche, 11836170001)

Note
This buffer is prepared at a 5X stock to achieve a final concentration of 1X in the resuspension buffer (4 X Buffer A + 1 X Buffer B).



Buffer C:

AB
HEPES pH 7.450 mM
Sodium Chloride150 mM
Magnesium Chloride5 mM
DTT0.5 mM
GDP100 nM
Sodium Fluoride50 mM
Sodium Pyrophosphate5 mM
Microcystin-LR (Enzo Life Sciences, ALX-350-012)1 μg/ml
Triton X-1001% (v/v)
cOmpleteTMEDTA-free Protease Inhibitor Cocktail (added fresh before use, Roche, 11836170001)

  • ReagentGibco™ PBS pH 7.4Thermo Fisher ScientificCatalog #10728775

  • ReagentPierce™ Coomassie Plus (Bradford) Assay KitThermo FisherCatalog #23236 or equivalent).

  • 4X Loading buffer:

ReagentNUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007 or 4X SDS loading buffer:

AB
Tris-HCl, pH6.8250mM
SDS8% (w/v)
Glycerol40% (v/v)
Bromophenol blue0.02% (w/v)
Reagents and antibodies:

Reagent Rubber tipped scraperMerck MilliporeSigma (Sigma-Aldrich)Catalog #CLS3008
ReagentAnti-Rab7 antibody Mouse monoclonalMerck MilliporeSigma (Sigma-Aldrich)Catalog #R8779
ReagentRecombinant Anti-RAB7 (phospho S72) antibody [MJF-R38-1] (ab302494)AbcamCatalog #ab302494
ReagentRecombinant Anti-PKC alpha antibody [Y124] (ab32376)AbcamCatalog #ab32376
ReagentRecombinant Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading ControlAbcamCatalog #ab76020
ReagentAnti-Rab7 antibody Mouse monoclonalMerck MilliporeSigma (Sigma-Aldrich)Catalog #R8779
Reagentα-Tubulin AntibodyCell Signaling TechnologyCatalog #2144
ReagentPhospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) AntibodyCell Signaling TechnologyCatalog #9101

Equipment:
Equipment
Corning® cell lifter
NAME
Corning® cell lifter
BRAND
CLS3008
SKU
https://www.sigmaaldrich.com/IN/en/product/sigma/cls3008
LINK
Blade L 19 mm, handle L 180 mm, sterile, case of 100
SPECIFICATIONS
or equivalent
Equipment
Eppendorf™ 5810R Centrifuge
NAME
Centrifuge
TYPE
Eppendorf
BRAND
02-262-8187
SKU
https://www.fishersci.com/shop/products/eppendorf-5810r-centrifuge-rotor-packages-16/022628187
LINK
or equivalent
Equipment
Eppendorf® microcentrifuge
NAME
Centrifuge
TYPE
Eppendorf®
BRAND
5417
SKU
https://www.sigmaaldrich.com/IN/en/product/sigma/z366013?gclid=CjwKCAjwtIaVBhBkEiwAsr7-cwVnwbWrBxOUW73qAEiKOBI00UeWVtRsRmiqBWQxzgNG-e_2iC6UNRoCZKAQAvD_BwE
LINK
or equivalent

Equipment
Luer Slip 1ml IV Syringes (Medicina IVS01)
NAME
Medicina Luer Slip IV Syringes can be used with any standard, filtered or safety needles.
TYPE
Medicina
BRAND
IVS01
SKU
https://medicina.co.uk/luer-slip-iv-syringes/
LINK
or equivalent
Equipment
25G Luer Needle (TerumoTM NN-2525R)
NAME
Terumo Hypodermic Needles
TYPE
Terumo
BRAND
NN-2525R
SKU
https://www.terumotmp.com/products/hypodermics/terumo-hypodermic-needles.html
LINK
or equivalent

Equipment
Thick-walled Polycarbonate Tubes (Beckman Coulter 343775)
NAME
Thick-walled Polycarbonate Tubes
TYPE
Beckman Coulter
BRAND
343775
SKU
https://www.mybeckman.in/supplies/tubes-and-bottles/open-top-tubes/343775
LINK
or equivalent


  • Ultracentrifuge (Beckman Coulter Optima TLX, or equivalent)

  • Ultracentrifuge rotor (Beckman Coulter TLS.55 or equivalent)

  • Plate reader for Protein quantification (BioTek Epoch, or equivalent)


Troubleshooting
Crude Membrane Fractionation

Note
The optimal quantity of cultured cells to use to achieve an ideal yield will vary dependent on cell type. As a guideline, we use 1 x 15cm dish of HEK293 cells per replicate seeded at 1.8 x 107 cells per dish.
Pour off media from the culture dish and aspirate completely by holding plate on edge. Wash cells twice with Amount5 mL of ice-cold PBS.
Pipetting
Wash
Immediately transfer the dishes to ice--this is best accomplished using wet paper towel-covered steel blocks resting TemperatureOn ice .
Add Amount5 mL of ice-cold PBS and scrape the cells from the dish using a cell lifter (Sigma-Aldrich CLS3008, rubber tipped scraper, or equivalent) to ensure good yield; collect in a 15 ml tube.
Pipetting
Pellet intact cells by centrifugation at Centrifigation100 x g for Duration00:05:00 at Temperature4 °C and aspirate supernatant.
5m
Centrifigation
Resuspend cells in Amount400 µL of Buffer A by gentle pipetting.
Pipetting
Transfer to an 1.5ml Eppendorf tube and incubate TemperatureOn ice for Duration00:15:00 .
Note
Note that this is a hypotonic solution and will swell the cells; Duration00:05:00 . is likely sufficient at this stage.


15m
Incubation
Pipetting
Add Amount100 µL of cold Buffer B to the cell suspension.
Pipetting
Using a 25-gauge needle attached to a 1 ml syringe, break the cells by passing the cell suspension through the needle 25 times.
Note
Breakage can be monitored by transferring a few microliters of the homogenate to a glass slide, covering with a coverslip and visualizing using a low power light microscope used to visualize cultured cells; as few as 6-10 passages may be sufficient. Broken cells will lose their reflective character and small particles of cell components will be readily detected.


Centrifuge the cell suspension at Centrifigation1000 x g for Duration00:05:00 at Temperature4 °C and collect the supernatant in a new 1.5ml Eppendorf tube.
Note
The pellet here will contain the nuclei and other cell debris. This can be analysed by lysing in Amount500 µL Buffer C. The supernatant represents the post-nuclear supernatant.


5m
Centrifigation
Pipetting
Load the post-nuclear supernatant into thick-walled polycarbonate tubes, appropriate for ultracentrifugation in a table top ultracentrifuge. Ultracentrifuge at Centrifigation150000 x g for Duration00:20:00 at Temperature4 °C .
Note
The membrane pellet will form at the bottom of the tube.

20m
Centrifigation
Transfer the cytosolic fraction (supernatant) to a fresh Eppendorf tube TemperatureOn ice .
Pipetting
Wash the membrane fraction pellet will Amount500 µL PBS thrice to remove any potential cytosolic contaminants.
Note
This may not be necessary if aspiration is complete.

Pipetting
Wash
Resuspend membrane pellet using Amount500 µL of Buffer C using a pipet and incubate TemperatureOn ice for Duration00:05:00 Duration00:20:00 to allow detergent solubilization of membrane proteins.
25m
Incubation
Pipetting
Centrifuge membrane protein solution at Centrifigation1000 x g forDuration00:05:00 at Temperature4 °C to separate solubilized membrane proteins (supernatant) from insoluble membrane proteins (pellet).
5m
Centrifigation
Determine the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate.
Note
Ensure the concentration of the samples is in the linear range for the Bradford assay. If it isn’t, prepare appropriate dilutions in water of each lysate. Generally, protein concentrations of near confluent cells lysed as described above should result in protein concentrations of at least Concentration2 µg/µL .


4×SDS–PAGE sample buffer is added to samples containing Amount5 µg of membrane protein or an equivalent volume of cytosolic protein, and heated at Temperature37 °C for Duration00:10:00 .
10m
Pipetting
Analysis of fractionation products by quantitative immunoblotting analysis
1d 16h 30m
The reaction products can be analysed by quantitative immunoblotting analysis (as described in dx.doi.org/10.17504/protocols.io.6qpvr68e3vmk/v1).

ABCDE
Antibody TargetCompanyCat. numberHost speciesDilution
pS72 Rab7AAbcam Inc.ab302494Rabbit1:1000
Rab7A (Total)SigmaR8779Mouse1:2000
LRRK1 (total) (C-terminus)MRC-PPU Reagents and Services, University of DundeeS405CSheep1 g/ml
TubulinCell Signaling Technologies2144Mouse1:5,000
pT202/Y204 ERK1/2Cell Signaling Technologies9101Rabbit1:1000
PKCαAbcam Inc.ab32376Mouse1:1000
Na-K ATPaseAbcam Inc.ab76020Rabbit1:10,000


Figure 1: Crude membrane fractionation of HEK293 Flp-in T-REx/GFP-LRRK1 WT cells following phorbol ester stimulation.
HEK293 Flp-in T-REx/GFP-LRRK1 WT cells were induced to express GFP-LRRK1 wild type by treatment with Concentration1 mg/mL doxycycline for Duration24:00:00 .
1d
Serum starve the cells for Duration16:00:00 and then treated ± Phorbol myristic acid (PMA) (Concentration100 ng/ml ) for Duration00:30:00 .
16h 30m
Following this, Perform the fractionation as described here and samples were subjected to immunoblot analysis with the indicated antibodies; the membranes were visualized using the Odyssey CLx scan Western Blot imaging system.
Note
Adapted from https://doi.org/10.1101/2022.06.09.495448.

Imaging