Hfq, Crc, and S1 Protein Detection

Equal amounts of total proteins were separated on a 12% SDS-polyacrylamide gel, and then electro-blotted onto a nitrocellulose membrane. The blots were blocked with 5% dry milk in TBS buffer, and probed with rabbit anti-Hfq, rabbit anti-Crc or rabbit anti-E. coli-S1 antibodies (Sonnleitner and Bläsi, 2014 (link)). Immunodetection of ribosomal protein S1 served as a loading control. The antibody–antigen complexes were visualized with horseradish peroxidase (HRP) conjugated anti-rabbit antibodies (Cell Signaling Technology) using the SuperSignal^TM West Pico PLUS chemiluminescent substrate kit (Thermo Scientific). The signals were detected with ChemiDoc^TM Touch Imaging System (BioRad) and analyzed with ImageLab 5.2.1 (BioRad).

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Sonnleitner E., Pusic P., Wolfinger M.T, & Bläsi U. (2020). Distinctive Regulation of Carbapenem Susceptibility in Pseudomonas aeruginosa by Hfq. Frontiers in Microbiology, 11, 1001.

Publication 2020

SuperSignalTM West Pico PLUS chemiluminescent substrate kit ChemiDocTM Touch Imaging System ImageLab 5.2.1

Anti antibodies Antibody antigen complexes Buffer E coli Horseradish peroxidase Membrane Milk Nitrocellulose Polyacrylamide gel Proteins Rabbit Ribosomal protein s1 Touch

Corresponding Organization : University of Vienna

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Variable analysis

independent variables

Rabbit anti-Hfq antibody
Rabbit anti-Crc antibody
Rabbit anti-E. coli-S1 antibody

dependent variables

Immunodetection of ribosomal protein S1

control variables

Equal amounts of total proteins
12% SDS-polyacrylamide gel
Nitrocellulose membrane
5% dry milk in TBS buffer
Horseradish peroxidase (HRP) conjugated anti-rabbit antibodies
SuperSignal™ West Pico PLUS chemiluminescent substrate kit
ChemiDoc™ Touch Imaging System
ImageLab 5.2.1 (BioRad)

controls

Immunodetection of ribosomal protein S1 as a loading control

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