Proteomic Analysis of Bacterial Regulatory Proteins

Cell pellets were collected, resuspended in FSB plus 0.2 M DTT, and boiled for fifteen minutes. At the time of loading, supernatants and cell pellets were normalized to the same number of cells. After separation on a 12.5% SDS-PAGE gel, proteins were transferred onto a blotting membrane (Immobilon-P) with a wet mini trans-blot cell (Bio-Rad). Blots were blocked for an hour in Tris-buffered saline with Tween 20 and 5% skim milk, and probed with the rabbit anti-RpoA (gift from Melanie Marketon), rabbit anti-LcrF (gift from Gregory Plano), rabbit anti-IscR [44 (link)], rabbit anti-YmoA (gift from Gregory Plano), rabbit anti H-NS (gift from Robert Landick), mouse M2 anti-FLAG (Sigma), goat anti-YopE (Santa Cruz Biotech), and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotech). Following visualization, quantification of the bands was performed with Image Lab software (Bio-Rad).

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Balderas D., Ohanyan M., Alvarez P.A., Mettert E., Tanner N., Kiley P.J, & Auerbuch V. (2022). Repression by the H-NS/YmoA histone-like protein complex enables IscR dependent regulation of the Yersinia T3SS. PLoS Genetics, 18(7), e1010321.

Publication 2022

Immobilon-P wet mini trans-blot cell mouse M2 anti-FLAG goat anti-YopE horseradish peroxidase-conjugated secondary antibodies Image Lab software

Antibodies Cell Goat Horseradish peroxidase Immobilon p Membrane Milk Mouse Pellets Proteins Rabbit Saline Sds page Tween 20

Corresponding Organization : University of Wisconsin–Madison

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Variable analysis

independent variables

Cell pellets were collected, resuspended in FSB plus 0.2 M DTT, and boiled for fifteen minutes.

dependent variables

Quantification of the bands was performed with Image Lab software (Bio-Rad).

control variables

At the time of loading, supernatants and cell pellets were normalized to the same number of cells.
After separation on a 12.5% SDS-PAGE gel, proteins were transferred onto a blotting membrane (Immobilon-P) with a wet mini trans-blot cell (Bio-Rad).

controls

Positive controls: None specified.
Negative controls: None specified.

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