Sucrose gradients

A continuous sucrose gradient IM - 2M can also been used, prepared either with a gradient maker or from di sion of a discontinuous gradient (sucrose concentrations IM to 2M with 2 or three intermediate concentrations, allowed to diffuse for several hr). 

Pickels (1943), Brakke (1951) and Kahler and Lloyd (1951) were the first few scientists to use stabilizing gradients. They used sucrose for these gradients. Sucrose still remains the material in most general use. 

Density gradients to stabilize flow have been employed by Philpot IT> Yin.s. Faraday Soc., 36, 38 (1940)] and Mel [ j. Phys. Chem., 31,559 (1959)]. Mel s Staflo apparatus [J. Phys. Chem., 31, 559 (1959)] has liquid flow in the horizontal direction, with layers of increasing density downward produced by sucrose concentrations increasing to 7.5 percent. The solute mixture to be separated is introduced in one such layer. Operation at low electrolyte concentrations, low voltage gradients, and low flow rates presents no cooling problem. 

EC 1.9.3.1]. Purified by selective solubilisation with Triton X-1(X) and subsequently with lauryl maltoside finally by sucrose gradient centrifugation [Li et al. Biochem J 242 417 1978]. 

Sucrose gradients for separation of membrane proteins must be able to separate proteins and protein-lipid complexes having a wide range of densities, typically 1.00 to 1.35 g/mL. 

The purification is done by sucrose density-gradient centrifugation (DeSa and Hastings, 1968 Hastings and Dunlap, 1986). Six sucrose... 

The stoichiometry of the recharged DNA/PLL/SPLL particles was studied using sucrose-gradient ultracentrifugation of fluorescently labeled polyion complexes in 25 mM HEPES buffer. Rhodamine-labeled DNA (Rh-DNA) and either fluorescein-labeled PLL (Fl-PLL) or SPLL (Fl-SPLL) were used to determine their relative amounts within DNA... 

Figure 7.26 Gradient-enhanced TOCSY spectrum of 10 mM sucrose in D. O recorded at 400 MHz by using modified MLKV-16 pulse sequence.

Gradient-enhanced 2D TOCSY spectrum of 10 mMof sucrose in D..,0 is shown in figure 7.26. The clean spectrum obtainable without any noise and without the nece.ssity of any phase cycling illustrates the power of this new technique in modern NMR spectroscopy. 

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). 

Sphaeroplasts were prepared by slight modifications to published methods [12,13]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

ATPase also catalyzed a passive Rb -Rb exchange, the rate of which was comparable to the rate of active Rb efflux. This suggested that the K-transporting step of H,K-ATPase is not severely limited by a K -occluded enzyme form, as was observed for Na,K-ATPase. Skrabanja et al. [164] also described the reconstitution of choleate solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. With the use of a pH electrode to measure the rate of H transport they observed not only an active transport, which is dependent on intravesicular K, but also a passive H exchange. This passive transport process, which exhibited a maximal rate of 5% of the active transport process, could be inhibited by vanadate and the specific inhibitor omeprazole, giving evidence that it is a function of gastric H,K-ATPase. The same authors demonstrated, by separation of non-incorporated H,K-ATPase from reconstituted H,K-ATPase on a sucrose gradient, that H,K-ATPase transports two protons and two ions per hydrolyzed ATP [112]. 

Siitterlin U, Thies W-G, Haffher H, et al. 1984. Comparative studies on the lysosomal association of monomeric 239Pu and 241Am in rat and Chinese hamster liver Analysis with sucrose, metrizamide, and percoll density gradients of subcellular binding as dependent on time. Radiat Res 98 293-306. 

Southern hybridization experiments, employing the cloned PHA synthase structural gene of Acinetobacter sp. and sucrose gradient fractions of DNA preparations separated in plasmid and chromosomal DNA fractions gave two hybridization signals and revealed some but not yet conclusive evidence for... 

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