Western Blot Analysis of Bacterial Proteins

Bacteria pellets were resuspended in PBS with 1X Final Sample Buffer (FSB) and, after boiling at 100 °C, loaded on 12.5% SDS-PAGE. A protein molecular weight marker (Page ruler; Thermo Fisher) was included in each electrophoresis run. Proteins were transferred to nitrocellulose membranes (Hybond-P, Millipore), and the immunoblotting was carried out with Monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich) and rabbit polyclonal anti-OmpA^{53 (link),54 (link)} as primary antibodies, and HRP conjugated goat anti-mouse and anti-rabbit IgG antibody as the secondary antibodies (Sigma-Aldrich). Signals were produced with ECL Star (Euroclone) and detected with ChemiDoc™ Gel Imaging System (Bio-Rad Laboratories). The densitometric analysis was performed by ImageJ software^{55 (link)}.

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Trirocco R., Pasqua M., Tramonti A., Colonna B., Paiardini A, & Prosseda G. (2023). Diffusible signal factors (DSFs) bind and repress VirF, the leading virulence activator of Shigella flexneri. Scientific Reports, 13, 13170.

Publication 2023

Page ruler Hybond-P Monoclonal ANTI-FLAG® M2 antibody ECL Star ChemiDoc™ Gel Imaging System

A protein Anti antibodies Anti antibody Antibodies Bacteria Buffer Densitometric Electrophoresis Goat Igg antibody Membranes Molecular marker Monoclonal antibody Mouse Nitrocellulose Pellets Proteins Rabbit Sds page

Corresponding Organization :

Other organizations : Istituto Pasteur, Sapienza University of Rome, National Research Council, Institute of Molecular Biology and Pathology

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Variable analysis

independent variables

Resuspension of bacteria pellets in PBS with 1X Final Sample Buffer (FSB)

dependent variables

Protein expression levels detected by immunoblotting with anti-FLAG and anti-OmpA antibodies

control variables

Protein molecular weight marker (Page ruler; Thermo Fisher) included in each electrophoresis run
Boiling of samples at 100 °C before loading on 12.5% SDS-PAGE
Protein transfer to nitrocellulose membranes (Hybond-P, Millipore)
Use of Monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich) and rabbit polyclonal anti-OmpA antibody as primary antibodies
Use of HRP conjugated goat anti-mouse and anti-rabbit IgG antibody as secondary antibodies (Sigma-Aldrich)
Signal detection with ECL Star (Euroclone) and ChemiDoc™ Gel Imaging System (Bio-Rad Laboratories)
Densitometric analysis performed by ImageJ software

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