Hcp Western Blot Analysis of Bacterial Cultures

Hcp western blots were performed as previously described^{34 (link)}. Briefly, supernatants and whole cell samples were obtained from mid-log bacterial cultures. Samples were resuspended in 1X Laemmli buffer and loaded in a 12% polyacrylamide gel for separation. The mouse anti-Hcp^{54 (link)} and rabbit anti-RNA polymerase (#663104, Biolegend, San Diego, CA) were both used at a concentration of 1:1000. Secondary IR dye antibodies from Licor were used at 1:10,000 (IRDye 800 CW goat anti-rabbit 926-32211 and IRDye 680 RD goat anti-mouse 925-68070). All blots were blocked in TBS blocking buffer (Licor).

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Di Venanzio G., Flores-Mireles A.L., Calix J.J., Haurat M.F., Scott N.E., Palmer L.D., Potter R.F., Hibbing M.E., Friedman L., Wang B., Dantas G., Skaar E.P., Hultgren S.J, & Feldman M.F. (2019). Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes. Nature Communications, 10, 2763.

Publication 2019

IRDye 800 CW goat anti-rabbit 926-32211 TBS blocking buffer

Antibodies Bacterial Buffer Cell Goat Irdye 800 Laemmli buffer Mouse Polyacrylamide gel Rabbit Rna polymerase Western blots

Corresponding Organization :

Other organizations : Washington University in St. Louis, Peter Doherty Institute, University of Melbourne, Vanderbilt University Medical Center, University of Buenos Aires

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Hcp protein levels (measured by western blot)

control variables

Bacterial culture conditions (mid-log phase)
Gel electrophoresis conditions (12% polyacrylamide gel)
Antibody concentrations (1:1000 for primary antibodies, 1:10,000 for secondary antibodies)
Blocking buffer (TBS blocking buffer)

controls

Positive control: RNA polymerase (detected by rabbit anti-RNA polymerase antibody)
Negative control: Not explicitly mentioned

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