Procedure

Mix 110-190 xg Chl PS2 (dependent on activity, see above) and 180 l liposome suspension add buffer to a final volume of 900 l. 2. Add 1 M OGP stock solution in small parts under mild stirring until the milky suspension is clear (start with 66 l 1 M OGP 2 OGP final conc. if still cloudy, add in steps of 0.5 ) 3. Add buffer up to a final volume of 1 ml and stir slowly in the dark at 20 C. 4. Add 80 mg Biobeads and incubate for 1 h repeat this step. 5. Add 160 mg Biobeads and incubate again for 1...

Setting up the microscope for bright field microscopy

Set up the microscope on a clean, dry area of bench as vibration-free as possible. 2. Plug in and switch on the illuminator. 3. Centre and focus the light source according to the manufacturer's instructions. The light source on many microscopes is precentred. 4. Bring the x10 objective into position on the nosepiece and ensure the condenser is in the bright field position, usually indicated by a zero (0). 5. Fully open the field diaphragm on the light source and the aperture diaphragm of the...

Comment

It is necessary to rule out the possibility of neuraminidase coming out from the vesicles as a result of sphingomyeli-nase activity. Ceramides increase membrane permeability, and efflux of molecules up to 40 kDa has been observed in sphingomyelin-treated vesicles 13 . Neu-raminidase has a molecular mass of 70 kDa. In order to clarify this aspect, a control experiment can be performed in which any extravesicular neuraminidase would be neutralized by a specific antibody. With this aim, a...

Plasma membrane see Protocols 425 and 426

As with ER and Golgi, the plasma membrane is often analysed along with other membrane compartments such as endosomes and the trans-Golgi network (TGN) during studies into membrane trafficking and cell signalling (see Chapter 5). Protocols 4.25 and 4.26 in this chapter, by contrast, are concerned primarily with the bulk (preparative) isolation of these membranes. There are two principal sources for bulk isolation of mammalian plasma membrane that have been used for studies of membrane...

Setting up the microscope for epifluorescence

The alignment of the light source to achieve an evenly illuminated field is critically important in epifluorescent microscopy 1. Turn on the lamp and allow to warm up. 2. Insert the filter block to be used. 3. Place a white piece of paper on the stage and adjust the light intensity to a suitable level with the slider. Open the condenser diaphragm and the field diaphragm. 4. Adjust the lamp collector focusing until a sharply focused image of the lamp is seen. Centre the image with the lamp...

Purification and preparation of spin labelled vimentin

Spin label compound (O-87500, methyl) methanethiosulfonate MTSL , Toronto Research Chemicals, Toronto, Canada) TCEP (tris-(2-carboxylethyl) phosphine, Molecular Probes, Eugene, OR) 1. Dissolve IBs (from 500 ml of bacterial culture) in 8 ml of 8 M urea (20 mM Tris pH 8, 1 mM EDTA, 8 M urea) and filter through a 0.2 micron filter (Pall Serum Acrodisc, Fisher Scientific). 2. Chromatograph 4 ml of inclusion body solution on a Hi Load 16 60 Superdex 200 column. The column is run with 20 buffer B,...

Setting up the microscope for phase contrast microscopy

The objectives for phase microscopy are different from those used for bright-field only. They include a phase ring and are usually inscribed Ph. Phase contrast objectives can also be used for bright field work. The condenser is also different it contains a series of phase annulae. 2. Kohler illumination is set up as described previously 3. To centre the phase contrast annular diaphragm, start with the objective x10 Ph 1. Set the condenser turret to the appropriate position. 4. Move the...