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Preparation of Membrane Rafts
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Preparation of Membrane Rafts

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Introduction Protocol References Credit lines
Category
Glycolipids and related compounds
Protocol Name

Preparation of Membrane Rafts

Authors
Inokuchi, Jin-ichi
Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University
KeyWords
Reagents

Phosphate buffered saline (PBS), pH 7.4, precooled on ice.

Detergent lysis (DL) buffer: 10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA, pH 7.4, and a suitable non-ionic detergent, such as 1% (v/v) Triton X-100 (TX-100), 0.5% (v/v) Lubrol WX, 0.5% (v/v) Brij-96, or 0.5% (v/v) Brij-98. An alternative to the DL buffer is an alkaline carbonate lysis buffer containing 100 mM sodium carbonate.

CompleteTM protease inhibitor cocktail tablets (Roche Applied Science, Penzberg, Germany) are added just prior to the start of the experiments at 1 tablet per 10 mL of buffer. We have found that this inhibitor cocktail is very effective in blocking proteases.

Instruments

A loose-fitting (e.g., Wheaton B pestle) 2-mL capacity Dounce homogenizer for cell disruption.

Probe sonicator (e.g., Sonics and Materials Vibra-CellTM VC 130 PB).

Round-bottom, polystyrene 5-mL tubes (e.g., FalconTM, BD, Franklin Lakes, NJ, Cat. No.352058)

Methods
1.

Glycolipid and related compounds

1) 

 Sucrose solutions for use in the density gradient: Sucrose (analytical grade) is added to 10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA, pH 7.4, to give final concentrations of 5%, 30%, and 80% sucrose solutions (all sucrose concentrations are w/v) that are required for the density gradient described here.

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2) 

 The cell homogenate treated with DL buffer (see the second item of Reagents and its comments in Notes) is pipetted into the bottom of a 12-mL ultracentrifuge tube (e.g., Beckman Coulter Ultra-ClearTM polycarbonate Cat. No. 344059). The volume is adjusted to 2 mL and then an equal volume of 80% sucrose is added. The two solutions are then mixed thoroughly by pipetting to give a final concentration of 40% sucrose in a 4-mL volume. This forms the bottom sucrose density layer in the ultracentrifuge tube.

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3) 

 4 mL of 30% sucrose solution are now layered on top using a 1-mL pipette. This has to be done slowly in order to avoid mixing with the 40% layer.

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4) 

 Finally, 4 mL of 5% sucrose are added and the tube is topped up with 5% sucrose to within a couple of millimeters of the rim. It is important to do this because even a 5-mm gap can result in the tube collapsing in on itself during ultracentrifugation. Performing this step next to the centrifuge will reduce the chance of spilling or disturbing the layers.

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5) 

 The tube is then carefully placed in a swing-out rotor and centrifuged overnight (or for at least 4 h) at 4°C at 175,000 × g.

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6) 

 After centrifugation, the swing-out buckets are carefully removed from the rotor. At this stage it may be possible to visualize a turbid white or opaque membrane band located between the 5% and 30% sucrose layers that contains the detergent-insoluble, low-buoyant density rafts.

Comment 1
7) 

 To harvest gradient fractions, a 1-mL pipette is used to slowly decant 12 1-mL fractions beginning at the top of the sucrose gradient. As the volume of the tube tends to be slightly greater than 12 mL, the 12th fraction tends to have a volume greater than 1 mL.

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8) 

 Protein assay: BCA (bicinchoninic acid) Protein Assay Reagent (Pierce Biotechnology).

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9) 

 Western blotting etc.

It is important to confirm that the membrane fraction containing lipid rafts contains markers reported in the literature. Western blotting can be used to confirm the presence of caveolin and flotillin. Conversely, Western blotting should also be used to demonstrate the absence of established non raft markers such as the transferrin receptor and clathrin.

Comment 1
Notes

This is the comment for the second item of Reagent :

In the method described, we suggest that an initial experiment should be designed around using the most commonly used detergent concentrations in the literature. However, for weakly raft-associated proteins, these concentrations may be too high and lead to their exclusion from the isolated lipid raft preparation and hence the misleading conclusion that they are not raft-associated. An example is the insulin receptor, which localizes to rafts prepared using lysis at low TX-100 concentrations (0.05%) but is lost at the standard 1% concentration5). Therefore, if, on the first round of analysis, a protein appears to not be raft-associated, repeating the experiment with reduced concentrations of detergent (>critical micelle concentration) in order to detect weaker raft interactions may be worthwhile. 

 

Discussion

The basic principle of the technique is to use a specific detergent to selectively solubilize non-raft regions of the membrane and to subsequently isolate the detergent-insoluble, low–buoyant density rafts by sucrose equilibrium density gradient centrifugation. The choice of detergent and concentration to use is a key issue6). However, it usually is not possible to predict either of these based solely on information such as the primary structure of the protein or its subcellular localization as determined by immunofluorescence or electron microscopy. Therefore, the idea is to comprehensively test a range of different detergents to establish which raft or raft subtype best defines the membrane domain to which the protein under investigation is targeted. There is no universally ideal single detergent, and results can be misleading: A given protein may be raft-associated in an intact cell (as determined by other techniques such as immunoelectron microscopy, fluorescence perturbation, or chemical cross-linking studies), but selectively solubilized from the raft by a detergent7) 8); conversely, a non-raft membrane protein or proteins present in different rafts may be inherently insoluble in a detergent and thereby left apparently co-localized in isolated detergent-insoluble rafts9) 10). The most commonly used detergents have been non-ionic detergents such as TX-100, Lubrol WX, Brij-58/97/98, and NP-40, each of which are selective for compositionally distinct cholesterol-rich membrane rafts6). As an example, Lubrol WX can be used to isolate cholesterol-sphingolipid lipid rafts that contain a high molar proportion of phosphatidylcholine, which is largely absent from TX-100 rafts11)

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