Aug 12, 2022
Crude Membrane Fractionation of Cultured Cells
- Asad Malik1,
- Dario R Alessi1,
- Suzanne R 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
External link: https://doi.org/10.1042/BCJ20220308
Protocol Citation: Asad Malik, Dario R Alessi, Suzanne R 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
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
Attachments
466-976.docx
781KB
Materials
MATERIALS
Reagents:
Buffer A:
10 millimolar (mM) HEPES 7.4 and cOmpleteTM EDTA-free Protease Inhibitor Cocktail
Note
Added fresh before use,cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
Buffer B:
| A | B | |
| HEPES pH 7.4 | 250 mM | |
| Sodium Chloride | 750 mM | |
| Magnesium Chloride | 25 mM | |
| DTT | 2.5 mM | |
| GDP | 500 nM | |
| Sodium Fluoride | 250 mM | |
| Sodium Pyrophosphate | 25 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:
| A | B | |
| HEPES pH 7.4 | 50 mM | |
| Sodium Chloride | 150 mM | |
| Magnesium Chloride | 5 mM | |
| DTT | 0.5 mM | |
| GDP | 100 nM | |
| Sodium Fluoride | 50 mM | |
| Sodium Pyrophosphate | 5 mM | |
| Microcystin-LR (Enzo Life Sciences, ALX-350-012) | 1 μg/ml | |
| Triton X-100 | 1% (v/v) | |
| cOmpleteTMEDTA-free Protease Inhibitor Cocktail (added fresh before use, Roche, 11836170001) | ||
- Gibco™ PBS pH 7.4Thermo Fisher ScientificCatalog #10728775
- Pierce™ Coomassie Plus (Bradford) Assay KitThermo FisherCatalog #23236 or equivalent).
- 4X Loading buffer:
NUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007 or 4X SDS loading buffer:
| A | B | |
| Tris-HCl, pH6.8 | 250mM | |
| SDS | 8% (w/v) | |
| Glycerol | 40% (v/v) | |
| Bromophenol blue | 0.02% (w/v) |
Reagents and antibodies:
Rubber tipped scraperSigma-aldrichCatalog #CLS3008
Anti-Rab7 antibody Mouse monoclonalSigma AldrichCatalog #R8779
Recombinant Anti-RAB7 (phospho S72) antibody [MJF-R38-1] (ab302494)AbcamCatalog #ab302494
Recombinant Anti-PKC alpha antibody [Y124] (ab32376)AbcamCatalog #ab32376
Recombinant Anti-Sodium Potassium ATPase antibody [EP1845Y] - Plasma Membrane Loading ControlAbcamCatalog #ab76020
Anti-Rab7 antibody Mouse monoclonalSigma AldrichCatalog #R8779
α-Tubulin AntibodyCell Signaling TechnologyCatalog #2144
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) AntibodyCell Signaling TechnologyCatalog #9101
Equipment:
Equipment
Corning® cell lifter
NAME
Corning® cell lifter
BRAND
CLS3008
SKU
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
LINK
or equivalent
Equipment
Eppendorf® microcentrifuge
NAME
Centrifuge
TYPE
Eppendorf®
BRAND
5417
SKU
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
LINK
or equivalent
Equipment
25G Luer Needle (TerumoTM NN-2525R)
NAME
Terumo Hypodermic Needles
TYPE
Terumo
BRAND
NN-2525R
SKU
LINK
or equivalent
Equipment
Thick-walled Polycarbonate Tubes (Beckman Coulter 343775)
NAME
Thick-walled Polycarbonate Tubes
TYPE
Beckman Coulter
BRAND
343775
SKU
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)
Crude Membrane Fractionation
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 5 mL of ice-cold PBS.
Immediately transfer the dishes to ice--this is best accomplished using wet paper towel-covered steel blocks resting On ice .
Add 5 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.
Pellet intact cells by centrifugation at 100 x g for 00:05:00 at 4 °C and aspirate supernatant.
5m
Resuspend cells in 400 µL of Buffer A by gentle pipetting.
Transfer to an 1.5ml Eppendorf tube and incubate On ice for 00:15:00 .
Note
Note that this is a hypotonic solution and will swell the cells; 00:05:00 . is likely sufficient at this stage.
15m
Add 100 µL of cold Buffer B to the cell suspension.
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 1000 x g for 00:05:00 at 4 °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 500 µL Buffer C. The supernatant represents the post-nuclear supernatant.
5m
Load the post-nuclear supernatant into thick-walled polycarbonate tubes, appropriate for ultracentrifugation in a table top ultracentrifuge. Ultracentrifuge at 150000 x g for 00:20:00 at 4 °C .
Note
The membrane pellet will form at the bottom of the tube.
20m
Transfer the cytosolic fraction (supernatant) to a fresh Eppendorf tube On ice .
Wash the membrane fraction pellet will 500 µL PBS thrice to remove any potential cytosolic contaminants.
Note
This may not be necessary if aspiration is complete.
Resuspend membrane pellet using 500 µL of Buffer C using a pipet and incubate On ice for 00:05:00 00:20:00 to allow detergent solubilization of membrane proteins.
25m
Centrifuge membrane protein solution at 1000 x g for00:05:00 at 4 °C to separate solubilized membrane proteins (supernatant) from insoluble membrane proteins (pellet).
5m
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 2 µg/µL .
4×SDS–PAGE sample buffer is added to samples containing 5 µg of membrane protein or an equivalent volume of cytosolic protein, and heated at 37 °C for 00:10:00 .
10m
Analysis of fractionation products by quantitative immunoblotting analysis
Analysis of fractionation products by quantitative immunoblotting analysis
1d 16h 30m
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).
| A | B | C | D | E | |
| Antibody Target | Company | Cat. number | Host species | Dilution | |
| pS72 Rab7A | Abcam Inc. | ab302494 | Rabbit | 1:1000 | |
| Rab7A (Total) | Sigma | R8779 | Mouse | 1:2000 | |
| LRRK1 (total) (C-terminus) | MRC-PPU Reagents and Services, University of Dundee | S405C | Sheep | 1 g/ml | |
| Tubulin | Cell Signaling Technologies | 2144 | Mouse | 1:5,000 | |
| pT202/Y204 ERK1/2 | Cell Signaling Technologies | 9101 | Rabbit | 1:1000 | |
| PKCα | Abcam Inc. | ab32376 | Mouse | 1:1000 | |
| Na-K ATPase | Abcam Inc. | ab76020 | Rabbit | 1:10,000 |
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 1 mg/mL doxycycline for 24:00:00 .
1d
Serum starve the cells for 16:00:00 and then treated ± Phorbol myristic acid (PMA) (100 ng/ml ) for 00:30:00 .
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.