Chloride gradients

Xylanase II was subjected to cation exchange chromatography with a 20-mm i.d., 700-mm long CM-Trisacryl column eluted with 0.8 mL/min of 0.025M sodium acetate buffer at pH 4.5, with a 0-0.2M sodium chloride gradient. Only one protein peak eluted from the column it contained all the Xylanase n activity. This material appeared... 

Chracteristics of B. m terium s plasmid system have been summarized by Carlton (3p. The plasmids are typical in that they band as covalently closed circular DNA in ethidium bromide-cesium chloride gradients and they are resistant to irreversible alkaline denaturation (17). However, B. megaterium plasmids are atypical in that they exist in approximately 10 size classes and as many copies per cell ( ). In fact, for the smaller plasmids there are hundreds of copies per cell so that plasmid DNA may represent up to 40% of the total extractable DNA (31). This is unusual since for most plasmids there are usually no more than a few copies per cell. Also, hybridization studies suggest that there is extensive homology between three B. megaterium plasmids of different sizes and between these plasmids and the chromosomal DNA (31,33). Carlton (31) concludes that the most likely explanation of the origin of B. megaterium plasmids is that they are molecular hybrids between one or more plasmid elements and various portions of the chromosomal DNA. 

If lithium chloride gradient is used, develop the sheet ascending for 2 min with Solvent 2, and then for 6 min with Solvent 2 and last up to 16 cm with Solvent 2". 

What is their volume If the mass of a bacterial ribosome is 2.7 x 106 daltons, what is its apparent density (divide mass by volume) Experimentally the buoyant density of bacterial ribosomes in a cesium chloride gradient (Chapter 5) is about 1.6 g/crn3. How can this difference be explained If eukaryotic ribosomes are 1.17 times larger than bacterial ribosomes in linear dimensions, what is the volume of a eukaryotic ribosome ... 

Taborsky (23) [see also Shapira (24)] has described a system based on carboxymethyl cellulose as the exchanger operated in Tris buffer at pH 8 with a sodium chloride gradient. The excellent and adjustable resolution of this system is frequently useful. The principal peak, labeled D by Taborsky, is indistinguishable from ribonuclease-A in the IRC-50 system (see Fig. 1). 

Following a wash step with 100 mL of buffer B, the ubiquitin was eluted by a sodium chloride gradient from 0 to 500 mM in purification buffer A (200 mL) (Fig. 1). The ubiquitin was eluted at about 150 mM of sodium chloride. 

Spin the tubes at 12,000 rpm for 10 min in an SS34 rotor and save the supernatant. At this point if multiple tubes for the same organism or sample have been prepared, the supernatants can be combined. Do not exceed the volume of the ultracentrifuge tubes to be used for the cesium chloride gradient, which is usually 8 ml. When all supernatants have been combined, if the volume is not 8 ml then adjust to 8 ml by adding TE. 

Once cell wall breakage or enzymatic dissolution has occurred, a variety of procedures can be used for isolation and purification of the DNA. These generally rely on an initial deproteinization using phenol or a mixture of perchlorate, sarcosine, and chloroform-isoamyl alcohol.6 9 The crude DNA is further purified by enzyme treatments, alcohol precipitation and spooling, hydroxylapatite chromatography, cesium chloride gradient centrifugation, or any combination of these methods. 

It is interesting to know that while the interaction between acidic proteins and hydroxyapatite is not sensitive to sodium chloride gradients, cationic proteins such as most of the antibodies, are eluted by a salt gradient. This is an advantage for their purification because all acidic contaminants... 

Fractionation is less specific and essentially requires separation efficiency. This is generally performed with ion-exchangers and hydrophobic interaction chromatography, which enable efficient separation by salt or pH gradients. Hydroxyapatite is also a good tool for fractionation where proteins can be separated by both phosphate gradients and sodium chloride gradients. 

Purification on CM HyperD F Adsorption at pH 5 Elution of adsorbed IgG with sodium chloride gradient ... 

An additional experiment that is most useful is the isolation of bacteriophage using cesium chloride gradients. Procedures for one such experiment involving the isolation of several varieties of phage are given in reference 8. 

Using Ecteola ion-exchange resin columns with acetate buffer and sodium chloride gradient from 0.06 to 0.22 M, Seijffers et al. eluted two... 

Fig. 2.4 Flowchart of the steps for rAAV production as described in Protocol 1. HEK293 cells are expanded, transfected, and harvested at 60h posttransfection. The cells are lysed, and loaded onto either a cesium chloride gradient or an iodixanol gradient to separate infectious virions from empty capsids. Virus is purified using either heparin affinity chromatography or ion-exchange chromatography. Virus preparations are formulated and concentrated, and characterized...

Cell Lysate and Cesium Chloride Gradient Protocol... 

Kornguth, S. E., Flangas, A. L., Siegel, F. L., Geison, R. L., O Brien, J. F., Lamar, Jr., C. and Scott, G. (1971) Chemical and metabolic characteristics of synaptic complexes from brain isolated by zonal centrifugation in a cesium chloride gradient. J. biol. Chem., 246, 1177-1184. 

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