Protein Detection Methods in Yeast

Total protein extracts were obtained from yeast cultures by trichloroacetic acid (TCA) preparation as described and were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto nitrocellulose membrane (33 (link)). Hemagglutinin (HA)-tagged Mec3 was visualized using a horseradish peroxidase (HRP)-conjugated anti-HA antibody (Roche, 3F10). Expression of genes cloned into the pBTM116 vector were detected with an anti-LexA antibody (Abcam, ab14553), and those cloned into the pACT2 vector were detected using an anti-HA antibody (Covance, 16B12). Myc-tagged Mcm10 was detected using an anti-Myc antibody (Thermo Scientific, 9E11), and endogenous Rad53 was detected using an anti-Rad53 antibody (gift from Dr J.F.X. Diffley).

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Alver R.C., Zhang T., Josephrajan A., Fultz B.L., Hendrix C.J., Das-Bradoo S, & Bielinsky A.K. (2014). The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage. Nucleic Acids Research, 42(13), 8389-8404.

Publication 2014

HRP)-conjugated anti-HA antibody anti-LexA antibody anti-HA antibody anti-Myc antibody

Anti antibody Expression genes Hemagglutinin Horseradish peroxidase Membrane Nitrocellulose Protein Sodium dodecylsulphate polyacrylamide gel electrophoresis Trichloroacetic acid Vector Yeast

Corresponding Organization :

Other organizations : University of Minnesota, Northeastern State University

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Variable analysis

independent variables

Cultures of yeast

dependent variables

Expression of HA-tagged Mec3
Expression of genes cloned into the pBTM116 vector
Expression of genes cloned into the pACT2 vector
Expression of Myc-tagged Mcm10
Expression of endogenous Rad53

control variables

Trichloroacetic acid (TCA) preparation for obtaining total protein extracts
Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation of proteins
Transfer of proteins onto nitrocellulose membrane
Use of HRP-conjugated anti-HA antibody for visualizing HA-tagged Mec3
Use of anti-LexA antibody for detecting expression of genes cloned into the pBTM116 vector
Use of anti-HA antibody for detecting expression of genes cloned into the pACT2 vector
Use of anti-Myc antibody for detecting Myc-tagged Mcm10
Use of anti-Rad53 antibody for detecting endogenous Rad53

controls

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

Annotations

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