Western Blot Analysis of GFP Expression

Total cellular protein extractions were carried out as described previously[53 (link)], and quantified using Bradford reagent (Bio-Rad), according to manufacturer’s instructions. A total of 50 μg of protein per sample was run on a 4% stacking and 12% resolving, self-made gel before being transferred to a membrane as described previously[14 (link)]. Blocking, primary (1:2000 dilution of the mouse monoclonal GFP antibody, #sc-9996, Santa Cruz Biotechnology) and secondary antibody (1:2000 dilution,[14 (link)]) incubations, and membrane revelation was carried out as described previously[14 (link)].

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Ries L.N., Pardeshi L., Dong Z., Tan K., Steenwyk J.L., Colabardini A.C., Ferreira Filho J.A., de Castro P.A., Silva L.P., Preite N.W., Almeida F., de Assis L.J., dos Santos R.A., Bowyer P., Bromley M., Owens R.A., Doyle S., Demasi M., Hernández D.C., Netto L.E., Pupo M.T., Rokas A., Loures F.V., Wong K.H, & Goldman G.H. (2020). The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence. PLoS Pathogens, 16(7), e1008645.

Publication 2020

Bradford reagent #sc-9996

A protein Antibody Cellular Dilution Membrane Monoclonal antibody Mouse Primary Protein

Corresponding Organization : Instituto Butantan

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Variable analysis

independent variables

Total cellular protein extractions

dependent variables

Protein expression levels

control variables

Bradford reagent (Bio-Rad) for protein quantification
4% stacking and 12% resolving, self-made gel for protein separation
Membrane transfer as described previously[14]
Blocking, primary (1:2000 dilution of the mouse monoclonal GFP antibody, #sc-9996, Santa Cruz Biotechnology) and secondary antibody (1:2000 dilution,[14]) incubations, and membrane revelation as described previously[14]

controls

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

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