Western Blot Analysis of A. fumigatus Proteins

Western blot analysis of A. fumigatus wild type and mutant protein levels was performed using a reported protocol (46 (link)). Briefly, lyophilized mycelial biomass was solubilized in NaOH, proteins were precipitated using trichloroacetic acid (TCA) and separated by SDS-PAGE using NuPAGE 4–12% (w/v) Bis–Tris gradient gels (Invitrogen). Proteins were transferred to a PVDF membrane using the iBlot 2 dry blotting system (Invitrogen). Western blots were reacted with rabbit α-HapX antiserum (1:20 000) and rabbit anti-β Actin (1:5000; abcam, ab119716) as primary antibodies and with peroxidase coupled anti-Rabbit IgG (1:10,000; Sigma, A1949) as secondary antibody. The membrane was developed using the 1-Step Ultra TMB-Blotting chromogenic substrate (Thermo Scientific).

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Furukawa T., Scheven M.T., Misslinger M., Zhao C., Hoefgen S., Gsaller F., Lau J., Jöchl C., Donaldson I., Valiante V., Brakhage A.A., Bromley M.J., Haas H, & Hortschansky P. (2020). The fungal CCAAT-binding complex and HapX display highly variable but evolutionary conserved synergetic promoter-specific DNA recognition. Nucleic Acids Research, 48(7), 3567-3590.

Publication 2020

NuPAGE 4–12% (w/v) Bis–Tris gradient gels iBlot 2 dry blotting system 1-Step Ultra TMB-Blotting chromogenic substrate

Actin Anti igg Antibodies Antibody Antiserum Bis tris Chromogenic substrate Gels Membrane Mutant protein Mycelial Peroxidase Proteins Pvdf Rabbit Sds page Trichloroacetic acid Western blots

Corresponding Organization :

Other organizations : University of Manchester, Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e. V. - Hans-Knöll-Institut (HKI), Innsbruck Medical University, Friedrich Schiller University Jena

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Variable analysis

independent variables

Wild type and mutant strains of A. fumigatus

dependent variables

Protein levels of target proteins

control variables

Lyophilized mycelial biomass solubilization in NaOH
Protein precipitation using trichloroacetic acid (TCA)
Separation of proteins by SDS-PAGE using NuPAGE 4–12% (w/v) Bis–Tris gradient gels
Transfer of proteins to a PVDF membrane using the iBlot 2 dry blotting system
Primary antibodies: rabbit α-HapX antiserum (1:20,000) and rabbit anti-β Actin (1:5,000)
Secondary antibody: peroxidase coupled anti-Rabbit IgG (1:10,000)
Membrane development using 1-Step Ultra TMB-Blotting chromogenic substrate

controls

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

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