Purification of DNA-origami nanostructures by rate-zonal centrifugation.

Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1-1 µg purified DNA nanostructures, obtaining >100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40-80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1-100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.

Isolation of renal brush borders.

Methods are described to isolate intact brush borders and brush border membranes from renal cell homogenates. A rapid method yields sealed vesicles that reconstitute renal brush border transport. In one variation of this protocol, 10 to 20 mM CaCl2 or MgCl2 is added to aggregate non-brush border structures for subsequent removal by centrifugation. For analytical studies, guidance is provided for subsequent purification steps including preparative free-flow and aqueous two-phase partition. Marker enzymes and morphological parameters are included for assessment of yield and fraction purity.

Purification, potency and immunogenicity analysis of Vero cell culture-derived rabies vaccine: a comparative study of single-step column chromatography and zonal centrifuge purification.

Continuous Vero cell lines are more suitable for large-scale production of rabies vaccine. The purification of Vero cell-derived rabies vaccine is critical because of the residual cellular DNA and serum proteins. The perfection of techniques using column chromatography with different matrix material, gel filtration and zonal centrifugation is of paramount importance for the optimal purification of rabies vaccine, leaving minimal residual cellular DNA, below the permissible level of 100 pg per dose and serum protein content of 1 ppm. In this study the potency, immunogenicity and safety of Vero cell-derived rabies vaccines were compared following purification by densely or loosely packed DEAE-sepharose CL-6B columns with different bed heights or by zonal centrifugation. The optimal virus recovery and maximum removal of substrate DNA and serum proteins were achieved only when the sepharose CL-6B column bed height was maintained at a thickness of 2-2.5 cm. The rabies virus material was purified by layering over the matrix without applying pressure. DEAE-sepharose CL-6B column purification using a simplified, cost effective technique as described in this study enhances the antigen yield by 50% in comparison with zonal purification.

Separation of membrane vesicles and cytosol from yeast, cultured cells, and bacteria in a small volume self-generated gradient in a fixed-angle rotor.

There are many situations when it is necessary to separate rapidly and efficiently a cytosolic and a membrane vesicle fraction from yeast, cultured cells, or from bacteria. This Protocol Article describes the flotation of the vesicles through a self-generated gradient from a dense sample zone using the low-viscosity medium iodixanol. As the sample is exposed to the gmax the tendency of the proteins to sediment overcomes any diffusion in the opposite direction and are therefore completely separated from the vesicles.

Overview of subcellular fractionation procedures for the yeast Saccharomyces cerevisiae.

This unit provides an overview of centrifugation-based fractionation procedures adapted for the yeast Saccharomyces cerevisiae. The goals, merits, limitations, and critical parameters of are discussed in order to facilitate the development of subcellular fractionation strategies. Topics include yeast cell lysate preparation, differential velocity centrifugation, density gradient centrifugation, and the analysis of subcellular fractions.

Determination of molecular size by zonal sedimentation analysis on sucrose density gradients.

The molecular weight of a protein is a basic characteristic that can only be approximated by techniques such as gel filtration and electrophoresis. Zonal sedimentation analysis on sucrose gradients is a method for estimating the molecular mass of proteins and protein complexes under nondenaturing conditions. This unit includes protocols for preparing the appropriate gradients, for fractionation and separation of cell lysates on the gradients, for fractionation of the gradients themselves, and use of the results to calculate the molecular mass based on sedimentation coefficient and other parameters. There is also an additional protocol for differential sedimentation on gradients made with water and deuterium oxide to allow for direct determination of the partial specific volume of a protein or complexes.

Separation of dorsal and ventral dopaminergic neurons from embryonic rat mesencephalon by buoyant density fractionation: disassembling pattern in the ventral midbrain.

The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine neurons, are organized into dorsal and ventral 'tiers' with regard to their ontogeny, efferent projections and their relative position in the various mesencephalic sub-nuclei. We have employed buoyant density fractionation to separate the dopaminergic neurons of the two compartments and compare their subsequent phenotype development with respect to their expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway. Using immunocytochemistry, separately and combined with in situ hybridization, we demonstrate here that sedimentation of cell suspensions from E19 rat ventral mesencephalon on 5-step Percoll gradients produces cell fractions enriched in ventral and dorsal tier DA neurons, respectively.

Biochemical characterization of the Golgi-complex proteins of Tritrichomonas foetus.

Using cell-fractionation techniques (differential and discontinuous gradient centrifugation), we obtained a highly enriched fraction containing the Golgi complex of Tritrichomonas foetus. This fraction was further subfractionated by sodium carbonate (150 mM) treatment at pH 11.5, leading to the isolation of the Golgi content and membrane subfractions. Both fractions were characterized by electron microscopy. The protein content of membrane and luminal subfractions was about 40% and 60% of the total Golgi protein, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed an enrichment of 15 protein bands in the Golgi fraction, with molecular masses varying between 15 and 116 kDa. Alkaline treatment released some proteins into the medium, but most of them were associated with the Golgi membrane.

The rate zonal separation of organelles from dilute suspensions: the problem of a large sample volume.

Transport vesicles that deliver proteins to the cell surface can be isolated by incubating cells that have been permeabilized by mechanical or chemical techniques in a high-K medium containing an ATP regenerating system. The vesicles released from permeabilized cells are, however, obtained as a very dilute suspension in the incubation solution. This presents a problem for the preparative separation of specific populations of vesicles by velocity sedimentation, because the small sample volume capacity of traditional glycerol or sucrose velocity gradients requires that the vesicles first be concentrated by sedimentation or that very small amounts of vesicles be loaded onto a gradient. We have addressed the problem of the loss of zonal resolution produced by the loading of large sample volumes, and we propose that high-viscosity Ficoll gradients can be used effectively to restore the resolution of zones when substantially larger sample volumes of dilute suspensions must be loaded onto velocity gradients.

A new method to measure iron absorption from the enrichment of 57Fe and 58Fe in young erythroid cells.

Iron absorption can be measured by the incorporation of stable iron isotopes into erythrocytes, 14 days after isotope administration. The disadvantage of this method is the high dose of isotopes needed to obtain a sufficient enrichment. Therefore, in this study cell fractions rich in young erythroid cells were prepared by using a density separation method. From 10 women blood was taken 4, 5, and 7 days after oral and intravenous administration of 57Fe and 58Fe. In these cell fractions and in whole blood taken 14 days after isotope administration, isotope enrichment was measured and absorption calculated. Absorption calculated from the isotope enrichment in the reticulocyte-rich cell fractions (12.2 +/- SEM 3.7%) was not significantly different from absorption based on whole-blood values (13.0 +/- 3.3%). Because a threefold higher isotope enrichment was found in the cell fractions, the required dose of stable isotopes can be reduced to one-third of the dose used in the traditional method without loss of sensitivity.

Rate zonal sedimentation of proteins in one hour or less.

Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein-protein interactions. However, rate zonal sedimentation experiments typically last approximately 1 day. In contrast, this report describes a rate zonal sedimentation method requiring 1 h or less. This was accomplished by centrifuging small density gradients (200 microliters) prepared with sucrose or OptiPrep in a fixed-angle rotor at high relative centrifugal force. By using small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. For a variety of proteins, plots of S20,w versus distance sedimented during centrifugation in a TLA 120.2 rotor were linear. As a practical application, sedimentation of the heterotrimeric stimulatory G protein and its dissociated alpha-subunit were determined. The results were similar to those obtained with 17- to 22-h centrifugations in an SW 50.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein-protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out traditional rate zonal sedimentation experiments. A rate zonal sedimentation technique that rivals many chromatographic methods in celerity will help to circumvent these problems.

Use of zonal centrifugation method for the preparation of mu-opioid receptor enriched membranes from bovine corpus striatum.

Earlier attempts to purify the opioid receptors met with limited success because of the use of brain crude membranes. Subcellular fractionation procedures adopted now to get enriched membranes resulted in 2-3 fold enrichment. However, a procedure has been developed in our laboratory in which a crude membrane fraction obtained at 17,500 x g was lysed with 1 mM sodium bicarbonate and later subjected to zonal fractionation, employing a density gradient of 0.6-1.2 M sucrose. This could yield a membrane fraction highly enriched with mu-opioid receptors which is 9.3 fold higher than the crude membranes.

Application of the hypo-osmotic swelling test to spermatozoa prepared by swim-up and discontinuous Percoll separation.

The quality of spermatozoa prepared by washing and swim-up or by discontinuous Percoll centrifugation, was compared by applying the hypo-osmotic swelling (HOS) test to semen samples from 116 men of infertile couples. The HOS test performed on 95 normal semen samples showed that the percentage of swollen spermatozoa separated by both techniques was significantly higher than in the initial ejaculate (p < 0.001). The percentage of HOS-positive spermatozoa separated by the Percoll gradient technique was significantly higher than that separated by the swim-up technique (p < 0.001). On the contrary, in 21 abnormal semen samples, there was no significant difference in the percentage of spermatozoa which were positive in the HOS test between the Percoll gradient and the swim-up technique (p = 0.44). It is suggested that the Percoll gradient technique appears to be preferable to the swim-up technique when semen parameters are normal, but there is no significant difference between these two technique in abnormal semen.