Back to article: Improvement of biochemical methods of polyP quantification

FIGURE 3: Ethanol precipitation method yields a broader spectrum of polyP sizes than affinity column purification.

(A) PAGE and DAPI staining of polyP differently purified. polyP was extracted using the neutral phenol/chloroform procedure from a yeast pellet equivalent to 107 logarithmically growing yeast cells. The aqueous phase was treated with DNAse and RNAse solution, and purified by affinity columns or by ethanol precipitation. The resulting polyP fractions: precipitated (in the case of the ethanol) and eluted and flow-through (in the case of the affinity column) were analyzed by PAGE followed by DAPI staining.

(B) Percentage of polyP relative to the purification method. polyP amount was determined using the same fractions obtained in panel A. The graph represents the Mean ± SEM from 3 independent experiments.

(C) Relative amount of polyP obtained after the precipitation of polyP in the presence of different monovalent salts, divalent salts and a carrier. Aqueous phase from panel A was used. The graph represents the percentage of polyP relative to precipitation with NaOAc. Mean ± SEM from 3 independent experiments is shown. GLC, glycogen.

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