Sucrose sedimentation alkaline

Douglas, GR., Blakey, D.H., Liu-lee, V.W., Bell, R.D.L. Bayley, J.M. (1985) Alkaline sucrose sedimentation, sister-chromatid exchange and micronnclens assays in CHO cells. 

Recent investigations using alkaline elution (51) or a combination of alkaline elution, alkaline sucrose sedimentation and... 

Function of DNA Ligase Some E. coli mutants contain defective DNA ligase. When these mutants are exposed to H-labeled thymine and the DNA produced is sedimented on an alkaline sucrose density gradient, two radioactive bands appear. One corresponds to a high molecular weight fraction, the other to a low molecular weight fraction. Explain. 

Alkaline sucrose centrifugation was introduced by McGrath and Williams,278 who showed that DNA from irradiated Escherichia coli sedimented more slowly than control DNA after lysis in alkali and that the sedimentation pattern approached that of the untreated DNA at increasing intervals after radiation treatment. Applying the procedure to mammalian cells, Lett and colleagues2 8 demonstrated rejoining of x-ray-induced strand breaks in the DNA of... 

Fig. 5. DNA strand breaks produced by a 30 min exposure of A549 cells to BLM (500 Mg ml ). Strand breaks were assayed by alkaline sucrose gradient centrifugation [14]. Sedimentation was from right to left. Immediately after BLM treatment O 2 h recovery, 0 mM 3AAB 2 h recovery, 5 mM 3AAB...

Although the enzyme sediments with intact cells, alkaline phosphatase appears in the supernate when broken cells are centrifuged. Malamy and Horecker (5) discovered that alkaline phosphatase is quantitatively released from the cell when E. coli are converted to spheroplasts by lysozyme and ethylenediaminetetraacetic acid (EDTA) in a sucrose medium. This evidence, supported by the observation that substrates such as glucose 6-phosphate are rapidly hydrolyzed by intact cells with release of most of the phosphate into the medium, led Malamy and Horecker (6) to suggest that alkaline phosphatase is localized in the periplasmic space, a region described by Mitchell (7) as lying between the protoplasmic membrane and the wall layer, and that it is not in association with the wall (8). 

To study the biochemical properties of membrane proteins, we use equilibrium sedimentation in a sucrose density step gradient (0.5 M, 2.0 M, 2.5 m) to purify the membrane fraction (Figure 32.1) following a procedure first developed by Strand et al. (1994). Protocol 32.1 describes this membrane-extraction procedure. Protocol 32.2 includes two useful methods to determine the membrane association of a protein of interest (1) alkaline wash and (2) Triton X-114 phase separation. The integrity of the Drosophila mem-... 

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