Yeast Protein Extraction and Analysis

Yeast protein extracts—except for chromatin fractionation—were prepared using the trichloroacetic acid protocol as described (18 (link)). For chromatin fractionation in Figure 5, total, soluble and pellet (chromatin) proteins were isolated as previously detailed for young yeast cells (35 (link)). Yeast proteins were run on an 8% (HA-Pds1, Brn1-Pk₉ and Pgk1), 6% (Myc-Top2 and Myc-Ubr1) or 15% (H4 and Pgk1) sodium dodecyl sulfate-polyacrilamyde gel. HA-Pds1, Myc-Top2, Myc-Ubr1, Pgk1, Brn1-Pk₉ and H4 were detected with the mouse monoclonal HA.11 (Covance), the mouse monoclonal 9E10 (Covance), the mouse polyclonal 22C5D8 (Invitrogen), the mouse monoclonal SV5-Pk1 (Serotec) and the rabbit polyclonal ab10158 (Abcam) antibodies, respectively. Peroxidase conjugate (Figures 1D, 4B, 4D and 5B) and fluorophore conjugate (Supplementary Figures S1A and S4A) secondary antibodies were used.

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Murillo-Pineda M., Cabello-Lobato M.J., Clemente-Ruiz M., Monje-Casas F, & Prado F. (2014). Defective histone supply causes condensin-dependent chromatin alterations, SAC activation and chromosome decatenation impairment. Nucleic Acids Research, 42(20), 12469-12482.

Publication 2014

HA.11 SV5-Pk1 ab10158

Antibodies Cells Chromatin Fractionation Mouse Peroxidase Proteins Rabbit Sodium dodecyl sulfate Top2 Trichloroacetic acid Yeast Yeast proteins

Corresponding Organization :

Other organizations : Consejo Superior de Investigaciones Científicas, Universidad de Sevilla

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Variable analysis

independent variables

Chromatin fractionation method

dependent variables

Total, soluble, and pellet (chromatin) proteins

control variables

Sodium dodecyl sulfate-polyacrilamyde gel concentrations
Antibodies used for protein detection (HA.11, 9E10, 22C5D8, SV5-Pk1, ab10158)
Peroxidase and fluorophore conjugate secondary antibodies

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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