Lipid Monolayer Assay Protocol

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pguo
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Lipid Monolayer Assay Protocol

 Background
Lipid monolayers have been used for many years as templates for the formation of two-dimensional crystals of soluble proteins (reviewed in (1)) and, more recently, membrane proteins (2). The principle of the assay is that phospholipids on an aqueous droplet adopt a conformation in which the hydrophobic tails point towards the air while the hydrophilic head groups contact the solution. Proteins of interest interact with the head groups and are concentrated in a two-dimensional array. A hydrophobic electron microscope grid interacts with the lipid tails, allowing the monolayer to be removed from the droplet and studied in the electron microscope. The lipid monolayer simulates the inner leaflet of the plasma membrane, and can be used to reconstitute early stages of endocytosis. The formation of ordered clathrin assemblies can be observed using negative stain electron microscopy, and platinum shadowing can reveal the invagination of these coats. For examples, see references (3,4). A schematic of the technique and a gallery of images obtained with this technique can be viewed using the links, or at http://www2.mrc-lmb.cam.ac.uk/groups/hmm/epsin/EM/
 
Reagents
HKM buffer (25mM Hepes pH 7.4, 125mM potassium acetate, 5mM magnesium acetate, 1mM dithiothreitol).
Chloroform
methanol
Phosphatidylinositol and Phospatidylinositol-4,5-bisphosphate (Avanti polar lipids) and were dissolved to 1mg/ml in 3:1 chloroform: methanol. Cholesterol (also Avanti) was dissolved to 10mg/ml in Chloroform. Phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine (all from Sigma) were dissolved in chloroform to 10mg/ml. Lipid stocks were stored at -80?C.
2% uranyl acetate (Biorad) with 0.0025% polyacrylic acid (Sigma) in water (see note 2)
Purified clathrin. Clathrin should be dialyzed into HKM buffer and centrifuged for 20 minutes at 100000 gmax (e.g., 45000 r.p.m. in a Beckman TLA100 rotor) immediately prior to use to remove aggregates.
Purified AP180, epsin, or other clathrin- and lipid-binding protein.

Equipment
Teflon block with 60 microliter wells allowing for side injection (see figure 1)
Carbon and collodion-coated gold electron microscopy grids (e.g., G204G from Agar Scientific, coated first with collodion or formvar, and then with a thin layer of evaporated carbon)
Humid chamber, or covered container with a wet paper towel inside
Forceps for handling EM grids—self-locking spring forceps are especially useful
Whatman filter paper or similar, for blotting EM grids.
Parafilm
Vacuum evaporator, 0.2mm diameter piece of platinum wire (TAAB Laboratories),1mm thick tungsten wire (also TAAB) (necessary for platinum shadowing)
Transmission electron microscope.

For the whole protocol, Refer to:

http://www.e-biotek.com/biochemistry.html