Vacuole Isolation and Cytosol Analysis

The cytosol was isolated from fermenting cells (pH 5) and filtered through a 10-kDa cutoff membrane as described (55 (link)). Filtered FTS was injected into a treated size-exclusion (SEC) Superdex Peptide 10/300 Gl column (GE Life Sciences) connected to an Agilent 1260 Bioinert quaternary pump (G5611A) and Agilent 7700x ICP-MS. The mobile phase was 20 mM ammonium acetate pH 6.5 flowing at 0.6 ml/min. Vacuoles were isolated as described (12 (link)).

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Kim J.E., Vali S.W., Nguyen T.Q., Dancis A, & Lindahl P.A. (2020). Mössbauer and LC-ICP-MS investigation of iron trafficking between vacuoles and mitochondria in vma2ΔSaccharomyces cerevisiae. The Journal of Biological Chemistry, 296, 100141.

Publication 2020

Superdex Peptide 10/300 Gl column Agilent 1260 Bioinert quaternary pump Agilent 7700x ICP-MS

Ammonium acetate Cells Cytosol Membrane Peptide Vacuoles

Corresponding Organization : Texas A&M University

Other organizations : University of Pennsylvania, Rutgers, The State University of New Jersey

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Variable analysis

independent variables

PH 5 of the cytosol
Filtration through a 10-kDa cutoff membrane
Injection of filtered FTS into a treated size-exclusion (SEC) Superdex Peptide 10/300 Gl column
Mobile phase of 20 mM ammonium acetate at pH 6.5 flowing at 0.6 ml/min

dependent variables

Unspecified

control variables

Unspecified

controls

Positive control: Vacuoles isolated as described in (12 (link))
Negative control: Not specified

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