Western Blot Analysis of Protein Expression

After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the iBlot (Invitrogen) dry blotting system. Polyclonal rabbit anti-StK and anti-StpA antibodies were used at recommended dilutions of 1/2,000 and 1/1,000, respectively (8 (link)). A Bis-Tris detergent-buffered saline solution with Hammersten casein was used for the block and antibody dilutions (Invitrogen; Western Breeze blocker/diluent). Goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Invitrogen) was used at a 1/10,000 dilution, and the proteins were detected with Clarity Western enhanced chemiluminescence (ECL) blotting substrate (Bio-Rad).

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Desbonnet C., Tait-Kamradt A., Garcia-Solache M., Dunman P., Coleman J., Arthur M, & Rice L.B. (2016). Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium. mBio, 7(2), e02188-15.

Publication 2016

iBlot Western Breeze blocker/diluent Clarity Western enhanced chemiluminescence (ECL) blotting substrate

Anti antibodies Anti antibody Antibody Bis tris Blotting western Casein Chemiluminescence Detergent Dilution Electrophoresis Goat Igg horseradish peroxidase Membranes Polyvinylidene difluoride Proteins Rabbit Saline solution

Corresponding Organization :

Other organizations : Brown University, Rhode Island Hospital, University of Rochester, Auburn University, Inserm, Sorbonne Université, Délégation Paris 5, Sorbonne Paris Cité, Université Paris Cité, Centre de Recherche des Cordeliers

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Variable analysis

independent variables

Antibody types (polyclonal rabbit anti-StK and anti-StpA antibodies)

dependent variables

Protein detection and visualization

control variables

Blotting system (iBlot dry blotting system)
Blocking and antibody dilution buffer (Bis-Tris detergent-buffered saline solution with Hammersten casein)
Secondary antibody (Goat anti-rabbit IgG horseradish peroxidase-conjugated)
Detection method (Clarity Western enhanced chemiluminescence (ECL) blotting substrate)

controls

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

Annotations

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