Sedimentation density gradient

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). 

In this approach a mixed solvent is chosen so that the relative sedimentation of the two components may give rise to a density gradient. The solute from a band that centres at the point where its effective density is equal to that of the solvent mixture. The band has a Gaussian shape with respect to solute concentration, the half-width is inversely proportional to the molecule weight of solute. A major importance is its sensitivity to small differences in effective density among the solute species. 

Regardless of the rotor speed and maximum velocity, sedimentation (or flotation) will not occur in a solution of equal density to the sample. Iso-density methods use this lack of movement in a manner comparable to a pH gradient in iso-electric focusing techniques. The methods are a combination of sedimentation and flotation, achieved by using a density gradient that straddles the density of the particles concerned. On centrifugation, the particles sediment until they reach a solvent zone with the same density. This results in the development of a zone for each type of particle present in the sample. 

RNA and DNA are isolated from tissues using caesium chloride density gradient sedimentation... 

The microsomal fraction was first obtained by Claude in 1943. In addition to lipid in the fraction, he noted the presence of RNA-rich granules, consistent with reports from Brachet that cytoplasm stained for RNA by the methyl-green/pyronin procedure. Glucose-6-phos-phatase was a prominent enzyme when the fraction was prepared from liver. Since density gradient sedimentation showed G-6-P-ase was absent from mitochondria and lysosomes, it was used as a marker for liver microsomes. 

Method Density gradient. Rate-zonal. The rate-zonal method is one of six addressed by SpinPro. The other methods are differential, differential-flotation, discontinuous, isopycnic, and 2-step isopycnic. These methods differ dramatically in their set up, principles of operation, and expected results. The rate-zonal method is described here briefly so that the recommendations to follow can be appreciated. Prior to the run in a rate-zonal method, a gradient material is introduced to the rotor tubes in steps of increasing density from the top to the bottom of the tube. The sample to be separated is layered, as a thin band, on the top of the gradient. As the run begins, each component in the sample moves toward the bottom of the tube. Some components sediment faster than others. This fact is the basis for the separation. If the run parameters are appropriate, the components will form separate bands within the gradient. At the conclusion of the run, the band representing the component of interest can be removed from the tube. 

Equilibrium sedimentation technique working with a multi-component solvent forming a density gradient in a centrifugal field... 

As a consequence of high osmolarity, macromolecules or organelles are dehydrated resulting in altered sedimentation behavior thus, different buoyant densities for nucleic acids and mitochondria are observed in different density gradient media, as illustrated in Table 5.3. Data of some density gradient media are given in Table 5.4. 

Leukocytes are prepared from EDTA-blood (approximately 3-5 ml) by the density gradient method. EDTA-blood is mixed by inversion and 5 ml is slowly added to 1 ml dextran solution. The blood and dextran solution are carefully mixed so that formation of foam is avoided. The mixture is allowed to stand for 1 h. If further time for sedimentation is required, it has to be noted as it may affect the resulting enzymatic activities. The time needed for proper sedimentation depends on sample quality and should not exceed 3 h. The upper phase including the white cells is transferred to another tube and spun at 600-1000 g for 10 min. The supernatant is... 

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. 

The sedimentation rate of the almond glucohydrolase on density gradient tests was slightly smaller than the rate for glucoamylase. Apparently the almond glucohydrolase is a somewhat smaller molecule. 

B 8. Assume that you have centrifuged in a density gradient a sample of DNA that contained both closed, circular DNA and supercoiled DNA Would you expect to see two bands in the sedimentation pattern 1 Explain. 

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