Western Blot Analysis of Bacterial Proteins

Bacterial pellets were resuspended in final sample buffer plus 0.2 M dithiothreitol (FSBS+DTT) and boiled for 15 min. At the time of loading, samples were normalized to the same number of cells by OD₆₀₀. Protein samples were run on a 12.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) and transferred to 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 rabbit anti-IscR (28 (link)), rabbit anti-YopD (gift from Alison Davis and Joan Mecsas), goat anti-YopE (Santa Cruz Biotechnology), rabbit anti-RpoA (gift from Melanie Marketon), and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotech). Gels were imaged by Image Lab software (Bio-Rad).

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Balderas D., Mettert E., Lam H.N., Banerjee R., Gverzdys T., Alvarez P., Saarunya G., Tanner N., Zoubedi A., Wei Y., Kiley P.J, & Auerbuch V. (2021). Genome Scale Analysis Reveals IscR Directly and Indirectly Regulates Virulence Factor Genes in Pathogenic Yersinia. mBio, 12(3), e00633-21.

Publication 2021

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

Antibodies Bacterial Buffer Cell Dithiothreitol Gels Goat Horseradish peroxidase Immobilon p Membrane Milk Pellets Polyacrylamide gel Protein Rabbit Saline Sodium dodecyl sulfate Tween 20

Corresponding Organization : University of California, Santa Cruz

Other organizations : University of Wisconsin–Madison, University of South Carolina

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Variable analysis

independent variables

Presence of dithiothreitol (DTT) in the final sample buffer

dependent variables

Protein expression levels of IscR, YopD, YopE, and RpoA

control variables

Number of cells used for protein sample normalization, as measured by OD600
Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) used for protein separation
Wet mini trans-blot cell (Bio-Rad) used for protein transfer to Immobilon-P membrane
Blocking solution (Tris-buffered saline with Tween 20 and 5% skim milk)
Primary antibodies (rabbit anti-IscR, rabbit anti-YopD, goat anti-YopE, rabbit anti-RpoA)
Horseradish peroxidase-conjugated secondary antibodies

controls

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

Annotations

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