Western Blot Analysis of Yeast Proteins

Protein was extracted from yeast cultures and analyzed by western blotting as previously described (43 (link)). Rabbit polyclonal anti-LexA (Invitrogen), rabbit polyclonal anti-Gal4_AD (Sigma) and mouse monoclonal anti-Pgk1 (Abcam) were used as primary antibodies with Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Novex) and anti-mouse IgG (Sigma) as secondary antibodies. Signals were detected by chemiluminescence using a Clarity Western ECL Substrate Kit (BioRad) and images captured using a VersaDoc 4000MP imaging system and Quantity One software (BioRad) or by autoradiography. Images were processed using Adobe Photoshop CS6 and figures prepared using Adobe Illustrator CS6.

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Mereshchuk A., Johnstone P.S., Chew J.S, & Dobson M.J. (2022). The yeast 2-micron plasmid Rep2 protein has Rep1-independent partitioning function. Nucleic Acids Research, 50(18), 10571-10585.

Publication 2022

mouse monoclonal anti-Pgk1 Horseradish peroxidase ( anti-mouse IgG Clarity Western ECL Substrate Kit VersaDoc 4000MP imaging system Quantity One software Illustrator CS6

Anti igg Antibodies Autoradiography Chemiluminescence Goat Horseradish peroxidase Mouse Protein Rabbit Yeast

Corresponding Organization : Dalhousie University

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Variable analysis

independent variables

Protein extracted from yeast cultures

dependent variables

Signals detected by chemiluminescence using a Clarity Western ECL Substrate Kit (BioRad) and images captured using a VersaDoc 4000MP imaging system and Quantity One software (BioRad) or by autoradiography

control variables

Rabbit polyclonal anti-LexA (Invitrogen), rabbit polyclonal anti-Gal4AD (Sigma) and mouse monoclonal anti-Pgk1 (Abcam) used as primary antibodies
Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Novex) and anti-mouse IgG (Sigma) used as secondary antibodies

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