Yeast Protein Extraction and Western Blot Analysis

Protein was extracted from yeast cultures using alkali lysis (12 (link)) and analyzed by western blotting as previously described (15 (link)). Antibodies used were rabbit polyclonal anti-Rep1 and anti-Rep2 (15 (link)), rabbit polyclonal anti-LexA (Pierce), mouse monoclonal anti-PGK (Molecular Probes), horseradish peroxidase-conjugated goat anti-rabbit (KPL) and Dylight 549 or 649-conjugated goat anti-mouse IgG (Rockland). Signals were detected by chemiluminescence using a Clarity Western ECL Substrate kit (BioRad) or by fluorescence and imaged using a VersaDoc 4000 MP imaging system with Quantity One software (BioRad) or by autoradiography.

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McQuaid M.E., Pinder J.B., Arumuggam N., Lacoste J.S., Chew J.S, & Dobson M.J. (2017). The yeast 2-μm plasmid Raf protein contributes to plasmid inheritance by stabilizing the Rep1 and Rep2 partitioning proteins. Nucleic Acids Research, 45(18), 10518-10533.

Publication 2017

Clarity Western ECL Substrate kit VersaDoc 4000 MP imaging system

Alkali Anti igg Antibodies Autoradiography Chemiluminescence Fluorescence Goat Horseradish peroxidase Molecular probes Mouse Protein Rabbit Yeast

Corresponding Organization : Dalhousie University

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Variable analysis

independent variables

Protein extraction method (alkali lysis)

dependent variables

Protein levels (as measured by Western blotting)

control variables

Yeast cultures
Western blotting protocols (as previously described)
Antibodies used (rabbit polyclonal anti-Rep1, anti-Rep2, rabbit polyclonal anti-LexA, mouse monoclonal anti-PGK, horseradish peroxidase-conjugated goat anti-rabbit, Dylight 549 or 649-conjugated goat anti-mouse IgG)
Signal detection methods (chemiluminescence, fluorescence, autoradiography)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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