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It is valid to think of the fractionations following polyploidy in the Brassicas (and probably following other plant polyploidies as well) as deletion machines ideally suited to be used in the procedure of fractionation mutagenesis.
A detailed protocol outlining the procedure of fractionation has now been accepted for publication and is currently in print in Nature Protocols: Depner, H., Lützkendorf, J., Babikir, H.A., Sigrist, S.J., and Holt, M.G. (2014): Differential centrifugation-based biochemical fractionation of the Drosophila adult CNS.
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The tar radical can be extracted into tert-butylbenzene and other organic solvents, and we have applied a variety of fractionation procedures to these solutions.
This may suggest that the fractionation procedure of the PSD does not disturb protein-protein interactions.
Alternatively, it may be the case that only more robust protein-protein associations survive during the fractionation procedure of the PSD, although the selective process is still consistent among samples.
As can be seen in Figure 6, for moderate rearrangement distances, and for a wide range of fractionation rates, our procedure almost completely wipes out the distortion caused by the fractionation.
Employing this procedure for subcellular fractionation of a crude organelle preparation, we obtained a fraction of intact vesicles that is significantly enriched in SL marker proteins.
The loss ratio of fractionation.
Pectin fractionation was carried out following the procedure of Rose et al. (1998) [ 31].
The procedure for membrane fractionation was essentially as described for the Nycodenz fractionation, except for the homogenization buffer used (25 mM Tris pH6.8, 150 mM NaCl, 1 mM PMSF, 20 μg/μl of leupeptin and 1 μg/μl of aprotinin).
After having established the validity of our fractionation procedure, equal volume of the fractions enriched in lipid rafts (3 6) and those identified as detergent-soluble (9 12) obtained from unstimulated and stimulated cells, were pooled and immunoprecipitated with an anti-IgE mAb in order to investigate the redistribution of ubiquitinated receptor subunits (Figure 4B).
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