Sucrose sedimentation

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... 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Figure 10.1 Experimental schemes for microarray analysis. All experimental schemes start with a separation step of the cell lysate by velocity sedimentation in a sucrose gradient (top scheme). Collection of the desired fractions is assisted by a continuous ultraviolet (UV) reading of the gradient (an example of such UV reading is shown in each section). This allows determination of the sedimentation position of the 40S, 60S, 80S, and polyribosomal complexes (2,3, and more).Three general ways for fraction collection and analysis are presented (sections A, B, and C) (A) Collection of two fractions (free and polysomes) and direct comparison between them, with the free mRNA fraction labeled with green dye and the polysome fraction labeled with red dye. (B) Collection of two fractions and indirect comparison between them by utilizing an unfractionated reference RNA. (C) Collection of multiple fractions (four in this case), where each fraction is compared to an unfractionated reference sample. The blue arrows indicate the addition of spike-in RNA to each fraction and to the reference RNA.

A summary of the report follows The problem is to separate proteins. Furthermore, SpinPro should pay particular attention to the purity of the separation. The sample is not negatively affected by sucrose, has a sedimentation coefficient of 16 Svedbergs, and is in liquid form of 3 mL and a concentration of 1% w/w. The protein of interest should be placed 45% from the top of the gradient at the end of the run. Of the gradient concentrations 10-40% and 5-20%, the 10-40% is preferred by the investigator. There are no solvents in the sample that are harmful to the tubes. Finally, from the lab, SpinPro should use the L2-75B ultracentrifuge and the SW 41 Ti rotor, which does not require a speed derating due to its age. 

Particle class Protein Separation vs Concentration Separation Optimization criterion Purity Assoc/Dissoc in sucrose No Sedimentation coefficient 16.0 10-40% or 5-20% gradient 10-40 Sample form liquid/semi-solid Total sample volume (mL) 3.0 Sample concentration % w/w 1.0 Selected final location 45.0 Solvents No... 

Fig. 2. NAPI facilitates H2A, H2B release from nucleosomes that are on positively coiled DNA (A) but not negatively coiled DNA (B). The positively coiled DNA (6.0 kb) with a superhelical density of + 0.05 and negatively coiled DNA (6.0 kb) with a superhelical density of -0.05 were reconstituted with lysine, arginine-labeled histones H3, H4, H2A, H2B by NaCl dialysis from 2.0 M to 1.2 M to 0.6 M to 0.1 M NaCl over a 14 h period. The samples were incubated with NAPI at 35 °C for 5 min and applied to a 5-20% sucrose/100 mM NaCl/40 mM Tris, pH 7.8 gradient. After sedimentation at 200,000 X g for 5 h, fractions were collected and the distribution of DNA (bottom panel) was determined on agarose gel and the distribution of protein (top panel) on SDS-PAGE followed by fluorography. These data are unpublished observations (V. Levchenko and V. Jackson). The deg-H2A is degraded H2A in which a 15 amino acid peptide of the C terminal has been proteolytically removed. When H2A, H2B is no longer present in a nucleosome, the C terminal region is sensitive to proteolysis [126] from a protease which is a minor contaminate in the NAPI preparation.

Variations in ferritin iron cores include the number of iron atoms, composition, and the degree of order (3,6,21-23). Size variations of the iron core range from 1-4500 Fe atoms and appear to be under biological control (e.g. Ref. 15). The distribution of iron core sizes in a particular ferritin preparation can be easily observed after sedimentation of ferritin through a gradient of sucrose. 

The sequence of steps in the biosynthesis of the ferritin iron core has been studied by analyzing the incorporation of Fe into ferritin during synthesis of the protein vivo. Ferritin, collected at various intervals after the induction of synthesis, was fractionated according to iron core size by sedimentation through gradients of sucrose (32). Fe appeared first in ferritin with small amounts of Fe, and later, the Fe appeared in fractions further down the gradient as the core size and the ratio... 

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