Immunoblotting Analysis of Yeast Proteins

Proteins were resolved by gel electrophoresis using NuPAGE Novex 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific). Proteins were transferred to nitrocellulose and bands detected by immunoblotting with specific rabbit polyclonal antisera. Antibodies against Sec61p, Sbh1p, CPY [29 (link)], Rpn12 [16 (link)], and preproalpha factor (ppαF) [29 (link)] had been previously raised in our laboratory and were used at a dilution of 1:2000; antibodies against Sss1p and Sec63p were kindly provided by Randy Schekman and used at a dilution of 1:2500. Signals were developed by enhanced chemiluminescence using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) according to the manufacturer´s instructions.

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Elia F., Yadhanapudi L., Tretter T, & Römisch K. (2019). The N-terminus of Sec61p plays key roles in ER protein import and ERAD. PLoS ONE, 14(4), e0215950.

Publication 2019

NuPAGE Novex 4–12% Bis-Tris Protein Gels SuperSignal West Dura Extended Duration Substrate

Antibodies Antisera Bis tris Chemiluminescence Dilution Dura Electrophoresis Gels Nitrocellulose Proteins Rabbit

Corresponding Organization : Saarland University

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Variable analysis

independent variables

Gel electrophoresis using NuPAGE Novex 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific)
Immunoblotting with specific rabbit polyclonal antisera

dependent variables

Resolution of proteins
Detection of protein bands

control variables

Nitrocellulose membrane for protein transfer
Enhanced chemiluminescence using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific)

controls

Positive controls: Antibodies against Sec61p, Sbh1p, CPY, Rpn12, and preproalpha factor (ppαF)
Negative controls: Not specified

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