Pelleted fraction

Fresh-frozen soluble fraction. d Fresh-frozen cell pellet fraction, n/a, not available IDs, identifications. 

A typical crude broken-cell preparation contains disrupted cell membranes, cellular organelles, and a large number of soluble proteins, all dispersed in an aqueous buffered solution. The membranes and the organelles can usually be separated from one another and from the soluble proteins by differential centrifugation. Differential centrifugation divides a sample into two fractions the pelleted fraction, or sediment, and the supernatant fraction, that is, the fraction that is not sedimented. The two fractions may then be separated by decantation. 

Dissolve the pellet with 5 pL of water. Identify both the supernatant and pellet fractions or both the total and the supernatant fractions by Coomassie blue staining of a 15-25% gradient SDS-PAGE gel (see Fig. 5). Verify that yeast ubiquitin is synthesized and is in the supernatant fraction. 

Mortar and pestle, blenders and homogenisers, grinders and sonicators Ammonium sulphate/acetate, salting out Sub-cellular fractionation in a centrifuge, up to 250 OOOg, supernatant and pellet fractions... 

Pilskaln C. A. and Honjo S. (1987) The fecal pellet fraction of biogeochemical particle fluxes to the deep sea. Global Biogeochem. Cycles 1(1), 31-48. 

In another aspect of the SODAS design, the secondary pellet fraction is produced as described above however, the delayed release beads are produced by coating the IR beads with an enteric polymer (methacrylic acid copolymer) rather than a sustained release polymer (81). This bi-modal release system was used in the formulation of Ritalin LA and was demonstrated to produce two distinct pla.sma concentration peaks separated by approximately 4 hours (81). This bi-modal pulsed release system was demonstrated to be bioequivalent to two immediate release tablet doses administered four hours apart. 

In comparison to the pellet fraction, the CPT in the supernatant fraction was much less sensitive to inhibition by 0.4 mM malonyl-CoA as it can be seen from the residual activity of about 88% but the sensitivity to L-AC was increased. At lOOpM L-AC the residual activity of CPT was about 20% indicating that a part of CPT 1 remained still in the supernatant probably due to incomplete sedimentation of mitochondrial membranes during the centrifugation. 

The Tween separation experiments add further evidence tsupporting this view. The mild detergent Tween 20 solubilizes selectively CPT II from mitochondrial inner membranes but keeps the CPT I bound at the mitochondrial outer membranes. By centrifugation it should be possible then to change significantly the ratio between both CPT-enzymes in the supernatant and the pellet-fraction. As shown in Fig. 2 this is obviously the case. In the supernatant the malonyl-CoA-insensitive fraction increased to 90% of total and decreased to 20% in the pellet fraction. 

Fig. 3 Transmission electron micrographs of ultrathin sections through a nuclei-enricbed pellet fraction isolated from rat livers. (A) Scale bar, 5 m (B) scale bar, 1 m.

Fig. 4 Transmission electron micrograph of an ultrathin section through a nudei-enriched pellet fraction isolated from baby chick (cockerel) livers. Scale bar, 5 urn.

Nuclei prepared by this alternative method can be recovered from the pellet fraction after centrifugation. At this point, they can either be used immediately... 

Figure 2 is a transmission electron micrograph of a nuclease-treated, Triton X-lOO-extracted, twice NaCl-extracted karyoskeletal protein-enriched fraction derived from Drosophila embryos. Nuclease treatment was at 37°C. Identifiable karyoskeletal elements are labeled. The SDS-PAGE profiles of the final karyoskeletal protein-enriched pellet fraction generated after subfractionation of nuclei treated with nucleases at 37°C are shown in Fig. 3 (lane 1) as well as the first 1 M NaCl extract generated after subfractionation of nuclei treated with nucleases at 23°C (also highly enriched for Drosophila karyoskeletal proteins but in soluble form) (lane 2). 

To appear in the pellet fraction after centrifugation, lamin must be assembled into relatively large aggregates (Lin and Fisher, 1990). Hence, the apparently abrupt transition in lamin behavior seen by microcentrifugation when mitotic/ meiotic lamin is mixed with interphase lamin at a ratio of 1 8 versus 1 4 (Fig. 3) does not, in our estimation, reflect an abrupt change in lamin polymerization. Rather, it reflects an operational peculiarity of the centrifugation assay. Centrifugation is nevertheless useful in that it is easily and rapidly performed. 

For disassembly, nuclei and oocyte extracts were thawed on ice immediately before use. Standard reaction mixtures were prepared on ice and included 2.7 mM ATP, 13.3 mM phosphocreatine, and 50 /ig/ml creatine phosphokinase. Reactions were incubated at 23°C, and at various times aliquots were removed and centrifuged at 4 C for 10 min at 12,000 g. Supernatant and corresponding pellet fractions were analyzed separately. For morphological analyses, samples were not fractionated but were fixed in 3.7% (w/v) formaldehyde and processed as described above. 

Biochemical analyses can be performed in parallel with or instead of morphological studies. At various time points during the course of in vitro disassembly, aliquots of the reaction mixture should be separated into supernatant and pellet fractions by sedimentation at 12,000 g and proteins subjected to SDS-PAGE and immunoblot analysis. An extract of stage 14 Drosophila oocytes contains only lamin Dm i, (Smith and Fisher, 1989 see also Lin and Fisher, 1990 Mans et at., 1995). Purified Drosophila nuclei contain lamins Dmi and Dm2 (Smith et al, 1987 see also Lin and Fisher, 1990 Maus et al, 1995). Lamin Dmmit migrates with a gel mobility intermediate to lamins Dm, and Dm2 (Fig. 4). 

To initiate disassembly, lamin aggregates (about 420 ng) assembled in vitro from affinity-purified lamins isolated from either embryo or p- S]methionine-labeled Kc nuclei should be mixed with 6.3 p. of oocyte extract in a final volume of 158 /a1. Samples should be separated into supernatant and pellet fractions after 90 min of incubation at 23°C and analyzed by SDS-PAGE and Coomassie blue staining or fiuorography as appropriate we chose to fractionate 75- t.l samples of the reaction described above. 

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