Dye precipitation
المؤلف:
Clive Dennison
المصدر:
A guide to protein isolation
الجزء والصفحة:
17-4-2016
1460
Dye precipitation
For the reaction:-
Protein in solution -------- Protein precipitate
most of the methods discussed above may be called “pushing” methods, in that the properties of the solution are changed, making these unsuitable for protein dissolution, i.e. the protein is pushed out of solution. If the protein is present at a very low concentration, in a large volume of solution, “pushing” methods can be quite uneconomical as the amount of precipitant required is proportional to the total solution volume, and often inversely proportional to the protein concentration. The alternative is a “pulling” method, in which properties of the protein are changed so that it comes out of solution. The push/pull terminology is due to Dr Rex Lovrien, of the University of Minnesota.
An example of a pulling method is dye precipitation or, as it has been called, “matrix co-precipitation”. Proteins are kept in solution by the disposition of charged, hydrophilic, groups on their surfaces. At low pH these are mainly positive and at high pH mainly negative. Dyes, on the other hand, are typically salts of strong acids or bases with attached aromatic groups having extended conjugation, which gives rise to their colour. If a dye having a negatively charged sulfonic acid group is added to a positively charged protein, ionic bonds will form between the dye and the protein. As a result, bulky, hydrophobic groups will become attached to the protein at its previously positive sites and the protein will be precipitated out of solution. An advantage of this method is that the amount of dye required is proportional to the moles of protein present, not to the volume of solution, so it is particularly suitable for harvesting proteins from dilute solutions.
After precipitation, it is necessary to separate the protein from the complexed dyestuff. This can be accomplished either by ion-exchange or by TPP. In TPP, the high salt concentration breaks the ionic bonds and the released dye is extracted into the t-butanol layer. Since dye precipitation is a pulling method and TPP is a “pushing” method, the sequential application of these two could be described as a “pull -push” method.
References
-Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .
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