TITLE: Crude Membrane Fractionation of Cultured Cells
AUTHORS: Asad Malik, Dario R Alessi, Suzanne R Pfeffer

[DESCRIPTION]

     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. 
 

 


[STEPS]
SECTION: Crude Membrane Fractionation
1. 
 
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.
SECTION: Crude Membrane Fractionation
2. Immediately transfer the dishes to ice--this is best accomplished using wet paper towel-covered steel blocks resting on ice.
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3. 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.
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4. Pellet intact cells by centrifugation at 100 x g for 5 min at 4 °C and aspirate supernatant.
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5. Resuspend cells in 400 µL of Buffer A by gentle pipetting.
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5.1. Transfer to an 1.5ml Eppendorf tube and incubate on ice for 15 min.
 

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6. Add 100 µL of cold Buffer B to the cell suspension.
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7.  Using a 25-gauge needle attached to a 1 ml syringe, break the cells by passing the cell suspension through the needle 25 times.
 

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8. Centrifuge the cell suspension at 1000 x g for 5 min at 4 °C and collect the supernatant in a new 1.5ml Eppendorf tube.
 

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9. Load the post-nuclear supernatant into thick-walled polycarbonate tubes, appropriate for ultracentrifugation in a table top ultracentrifuge. Ultracentrifuge at 150000 x g for 20 min at 4 °C. 
 

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10.  Transfer the cytosolic fraction (supernatant) to a fresh Eppendorf tube on ice.
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11. Wash the membrane fraction pellet will 500 µL PBS thrice to remove any potential cytosolic contaminants.
 

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12. Resuspend membrane pellet using 500 µL of Buffer C using a pipet and incubate on ice for 5 min  20 min to allow detergent solubilization of membrane proteins.
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13. Centrifuge membrane protein solution at 1000 x g for5 min at 4 °C to separate solubilized membrane proteins (supernatant) from insoluble membrane proteins (pellet).
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14. Determine the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate. 
 

SECTION: Analysis of fractionation products by quantitative immunoblotting analysis
16. The reaction products can be analysed by quantitative immunoblotting analysis (as described in dx.doi.org/10.17504/protocols.io.6qpvr68e3vmk/v1).

 

SECTION: Analysis of fractionation products by quantitative immunoblotting analysis
17. 
  
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 1440 min. 
SECTION: Analysis of fractionation products by quantitative immunoblotting analysis
18. Serum  starve the cells for 960 min and then treated ± Phorbol myristic acid (PMA) (100 ng/ml) for 30 min. 
SECTION: Analysis of fractionation products by quantitative immunoblotting analysis
19. 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. 
 

SECTION: Crude Membrane Fractionation
15. 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 10 min.