Immunoblotting analysis of Laz protein

Samples were normalized by OD₆₀₀ values (whole cell lysates), protein concentration (subcellular fractionation samples), or colony forming units (CFU/mL; murine vaginal washes). Proteins were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% Novex NuPAGE (Thermo Fisher Scientific), 4–15% Bio-Rad Criterion TGX (Bio-Rad), or Bio-Rad AnykD Criterion TGX (Bio-Rad) gels and visualized by colloidal Coomassie G-250 staining. Immunoblots were performed as described (Zielke et al., 2016 (link); Sikora et al., 2017 (link), 2018 (link); Wierzbicki et al., 2017 (link)). The polyclonal rabbit anti-Laz antiserum used in the current study was generated previously by immunization with recombinant His-tagged Laz (Zielke et al., 2016 (link)).

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Baarda B.I., Zielke R.A., Jerse A.E, & Sikora A.E. (2018). Lipid-Modified Azurin of Neisseria gonorrhoeae Is Not Surface Exposed and Does Not Interact With the Nitrite Reductase AniA. Frontiers in Microbiology, 9, 2915.

Publication 2018

Novex NuPAGE Bio-Rad Criterion TGX

Antiserum Cell Fractionation Gels Immunization Immunoblots Murine Proteins Rabbit Sds page Vaginal

Corresponding Organization : Oregon Health & Science University

Other organizations : Uniformed Services University of the Health Sciences

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Variable analysis

independent variables

Normalization method (OD600, protein concentration, or CFU/mL)

dependent variables

Protein expression levels (visualized by Coomassie G-250 staining and immunoblotting)

control variables

Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) parameters (4–12% Novex NuPAGE, 4–15% Bio-Rad Criterion TGX, or Bio-Rad AnykD Criterion TGX gels)

controls

Positive control: Polyclonal rabbit anti-Laz antiserum generated by immunization with recombinant His-tagged Laz
Negative control: Not explicitly mentioned

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