Purification and preparation of spin labelled vimentin

Nuclear Matrix

Spin label compound (0-87500, 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,...

EM methods available

The techniques used for biological specimen preparation fall into two main categories, those utilizing resin embedding followed by thin sectioning and positive staining or immunolabelling, and those using a thinly spread layer of particulate material, followed by metal shadowing, air-dry negative staining or rapid freezing vitrification without or with negative staining. The technique of freeze-fracture, although widely used in the past, is somewhat less popular today although it remains useful...

Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling

Protocol 5.1 Separation of basolateral and bile canalicular plasma membrane domains from mammalian liver in sucrose gradients 160 Protocol 5.2 Isolation of rat liver sinusoidal domain using Protocol 5.3 Fractionation of apical and basolateral domains from Caco-2 cells in a sucrose gradient 163 Protocol 5.4 Fractionation of apical and basolateral domains from MDCK cells in an iodixanol gradient 165 Protocol 5.5 Isolation of lipid rafts 167 Protocol 5.6 Isolation of caveolae 170 Protocol 5.7...

Key components of the compound microscope

Transillumination Knob Microscope

The eyepieces, body tube, nosepiece and objectives are part of the magnification system of the microscope. The condenser, condenser-iris diaphragm, filters, field iris diaphragm and light source are the parts that compose the illumination system of the microscope. To use a microscope properly, and to get the most out of it, it is important to understand the purpose and function of each of the microscope's components Figure 1.2 . The binocular body, the arm and the base form the frame of the...

Homogenization

For most purposes, an isoosmotic medium containing 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4 may be used for any soft tissue or cultured animal cell. There are a number of minor variations such as adding EGTA instead of, or in addition to, EDTA and substituting Tris by an alternative organic buffer Hepes or Tricine . Such modifications do not materially affect the result of the fractionation procedure they are normally introduced to be more compatible with some subsequent add-on...

Mechanical disaggregation of tissue

Mincing or chopping tissue in liquid medium releases cells from the supporting matrix the isolated cell population often includes large numbers of red blood cells and the viability of nucleated cells may be low as a result of mechanical trauma. However, most disaggregation procedures begin with a mechanical step to reduce the tissue to small fragments, providing a larger surface area for exposure to the digesting enzymes and facilitating penetration of enzymes into the tissue. Soft tissues can...

Nucleiand nuclear components see Protocols 4146 Nuclei

Detergent-free methods for the purification of nuclei have routinely involved the pelleting of the organelles from a crude nuclear pellet suspension, through a dense approx 2 M sucrose barrier. The nuclei lose water to the grossly hyperosmotic medium and attain a density gt 1.32 g ml. Pelleting the nuclei is thus the only option since this is the limiting density of sucrose solutions. The high viscosity of the solutions also requires a g-force, normally of 100 000g or higher, for at least 1 h....