A method for the isolation of rat submandibular salivary gland polysomes on linear sucrose density gradients

A procedure for isolating rat submandibular salivary gland polysomes on sucrose density gradients has been described. Electron micrographs of the gland revealed the existence of a very well-developed system of membranebound ribosomes. These morphological findings dictated the use of vigorous homogenization of tissue, a detergent, and low g-forces during tissue fractionation procedures for maximal recovery of this cellular organelle. The methodology allowed for the isolation of polysomes susceptable to RNase degradation and able to incorporate labeled amino acids into protein in a cell-free system.     

A simple technique for the preparation and storage of sucrose gradients

A method for preparing multiple sucrose gradients by quickly freezing layers of sucrose has been developed. These gradients may be stored in the freezer indefinitely, and thawed from 8 to 24 h at 4 degrees C before use. The middle region of the resulting sucrose gradients was linear. Thawing time and centrifugation had little effect on the shape of the gradient. The method is applicable for both small- and large-volume centrifuge tubes. Gradients prepared in the same batch were nearly identical.     

Clinical performance of a system for semiautomated chromosome analysis.

Until recently equipment for automated chromosome analysis has not been used for routine purposes in clinical cytogenetic laboratories. During a 3 1/2-year period the chromosome laboratory of Rigshospitalet has tested the Magiscan chromosome system under routine conditions and performed the first evaluation of its clinical performance. The system consists of an image processor with a light pen for manual interaction connected to a hard-copy printer and a microscope with a TV camera and a motorized scanning stage for eight slides. Automated metaphase finding takes place without operator assistance. An operator is involved in the analysis after the metaphases are located. Using two of these complete systems, we have performed a total of 4,691 chromosome analyses comprising a count of 10 metaphases, of which three were "eyeball" karyotyped and one was "machine" karyotyped. Presently, two-thirds of our prenatal analyses (amniotic-cell cultures) are carried out with these two machines. A third Magiscan system without scanning stage is used as a "karyotyping-only" system to produce hard-copy karyograms in those cases in which metaphases are manually located and counted in the microscope. Since the end of 1984, 4,773 additional machine karyograms have been produced with this system. With a complete system, a prenatal analysis can be carried out in an average of 35 min. The average time for a machine karyotype is 7 min. Since 1984 the productivity of the laboratory has increased 17%-20% without enlarging the staff.     

The construction and analysis of sucrose gradients for use with zonal rotors.

The rate of sedimentation of a particle in a sucrose solution depends on the viscosity and density of the medium. These two variables are related to the sucrose concentration and the temperature of the medium by new simple equations. These equations were used in a rapid iterative procedure that relates the distance moved by a zone in a continuous sucrose gradient to its sedimentation coefficient. It is shown by comparison with experiment that this iterative method allows the distance moved by a zone to be calculated rapidly. The method may therefore be used to optimize the separation of particles in a sucrose-gradient-centrifugation experiment. The method also allows the unknown sedimentation coefficients of several zones to be measured from a single sucrose-gradient-centrifugation experiment.     

A method for predicting the location of particles sedimenting in sucrose gradients

A theory was developed for the calculation of the positions of zones of particles sedimenting through a sucrose gradient. Equations were derived for particles sedimenting through gradients in which the sucrose concentration is (a) a linear function of radius, or (b) a hyperbolic function of radius. Computations were made for both swing-out and zonal rotors. The theory, which is based on direct integration of the sedimentation equation, exploits equations relating (a) the density of sucrose solutions to sucrose concentration and (b) the viscosity of sucrose solutions to sucrose concentration, and also the concept of reduced time (T/2 = S20.w integral of t to w2dt) of Fujita. The required computations may be made using a scientific calculator. Experimental support for the theory was obtained.     

A simplified method for preparing sucrose gradients (Short Communication)

Numerous sucrose gradients can be prepared simultaneously by diffusion in horizontal centrifuge tubes.     

Microvilli of the human term placenta. Isolation and subfractionation by centrifugation in sucrose density gradients.

Human placental microvilli were isolated and separated into two fractions by centrifugation in sucrose density gradients. Electron-microscopic morphology and morphometry, the distribution of enzymic activities and the results of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of proteins were used to assess the purity of the final preparations and to define their properties. The combined evidence strongly suggested that the preparations contained negligible material that was not plasma membrane. The two fractions of microvilli differed in buoyant density, protein composition, enzyme specific activities and microscopic appearance. Some of these differences were explained by the absence of internal structure in the microvilli of the lighter fraction.     

Vertical dye-buoyant density gradients for rapid analysis and preparation of plasmid DNA

A rapid and easy method for the isolation of plasmid DNA, both in analytical and preparative scale, is described. Using dye-buoyant density-gradient centrifugation in a vertical rotor, separation of covalently closed, supercoiled plasmid DNA from relaxed circular and linear DNA is completed within 1 to 2 h.     

Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion-free sucrose density gradients

A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.     

A semiautomated system for measurement of 96 simultaneous spectrophotometric enzyme assays

A semiautomated system for spectrophotometric measurement of enzyme activity is described. In comparison to a 1-ml reaction volume monitored continuously by a conventional spectrophotometer, this system requires 1/10 to 1/100 the volume of sample, and 1/8 to 1/4 the time for measurement and computation of 96 enzyme assays. The system hardware consists of a 96-well platereader interfaced to a personal computer. Absorbances of 96 reactions are measured at timed intervals. These data are transmitted electronically from the platereader to the computer through the modem port using a modem program. The reaction rates are computed from the timed absorbance readings using a spreadsheet program. Three enzyme assays are presented, but the method has been used for several other assays and is applicable to many spectrophotometric rate assays. Many laboratories currently possess one or both of the two major components of the relatively inexpensive system described.     

Isolation of protein bodies on sucrose gradients

Summary Storage protein bodies from sunflower cotyledons during early stages of seed germination were isolated on sucrose density gradients by isopycnic centrifugation. The density of this organelle on the gradients ranged between 1.26 and 1.36 g cm^-3. A proteinase with a pH optimum of 5.2 was associated with this organelle, and is probably responsible for degradation of storage protein. A NADH-dependent cytochrome-c reductase, a membrane marker enzyme with a pH optimum of 8.4, was also present in this organelle fraction.Abbreviations LPA for l-lysine-p-nitroanilide - LPAase for the peptidase which hydrolyzes this peptide This work was supported in part by the National Science Foundation Grant GB-17543, and published as Journal Article No. 5736 of the Michigan Agricultural Experiment Station.Supported by a Deutsche Forschungsgemeinschaft Fellowship.