Protein Expression Analysis via Western Blot

Western blot analyses of protein samples were performed as described previously for rabbit anti-Tse1 (diluted 1:5,000; Genscript), rabbit anti-FLAG (diluted 1:5,000; Sigma), rabbit anti-VSV-G (diluted 1:5,000; Sigma), rabbit anti-Hcp1 (P. aeruginosa) (diluted 1:5,000, Genscript) and detected with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (diluted 1:5,000; Sigma) (Ahmad et al., 2019 (link)). Rabbit anti-Hcp (P. protegens) was used at a 1:5000 dilution. Western blots were developed using chemiluminescent substrate (Clarity Max, Bio-Rad) and imaged with the ChemiDoc Imaging System (Bio-Rad).

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Ahmad S., Tsang K.K., Sachar K., Quentin D., Tashin T.M., Bullen N.P., Raunser S., McArthur A.G., Prehna G, & Whitney J.C. (2020). Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones. eLife, 9, e62816.

Publication 2020

rabbit anti-FLAG rabbit anti-VSV-G anti-rabbit horseradish peroxidase-conjugated secondary antibodies Clarity Max ChemiDoc Imaging System

Anti antibodies Dilution Horseradish peroxidase P aeruginosa Protein Rabbit Western blots

Corresponding Organization :

Other organizations : McMaster University, University of Manitoba, Max Planck Institute of Molecular Physiology, Discovery Centre

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Variable analysis

independent variables

Rabbit anti-Tse1 antibody dilution (1:5,000)
Rabbit anti-FLAG antibody dilution (1:5,000)
Rabbit anti-VSV-G antibody dilution (1:5,000)
Rabbit anti-Hcp1 (P. aeruginosa) antibody dilution (1:5,000)
Rabbit anti-Hcp (P. protegens) antibody dilution (1:5,000)

dependent variables

Protein expression levels detected by Western blot analysis

control variables

Western blot analysis protocol (as described previously)
Anti-rabbit horseradish peroxidase-conjugated secondary antibody dilution (1:5,000)
Chemiluminescent substrate (Clarity Max, Bio-Rad) for Western blot development
Imaging with ChemiDoc Imaging System (Bio-Rad)

controls

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

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