Samples were electrophoresed in 4–12% NuPAGE polyacrylamide gels (Invitrogen) or 10–20% Tris/glycine polyacrylamide gels (Bio-Rad Laboratories). Proteins were transferred to PVDF membranes (Bio-Rad Laboratories), and membranes were stained for 10 min with Coomassie blue (50% methanol, 10% acetic acid, and 0.05% Coomassie blue R-250 [MP Biomedicals]) and washed with 50% methanol to confirm transfer uniformity. Membranes were incubated with blocking buffer (StartingBlock), and then, 1.66 µg/ml anti-PMY mAb (12D10) and human anti–ribosomal P antibody (1:3,000) were added in buffer (StartingBlock) and incubated overnight at 4°C. After washing three times with wash buffer (PBS and 0.1% Tween 20), secondary antibodies were added in buffer (StartingBlock) and incubated for 1 h. Membranes were washed three times, and ECL substrate (SuperSignal; Thermo Fisher Scientific) was used to detect staining by exposure to x-ray film.
Protocol detail
Western Blot Analysis Procedure
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Acetic acid
Anti antibody
Antibodies
Buffer
Coomassie blue
Coomassie blue r 250
Glycine
Human
Membranes
Methanol
Polyacrylamide gels
Proteins
Pvdf
Ribosomal
Tris
Tween 20
X ray film
Corresponding Organization :
Other organizations : National Institute of Allergy and Infectious Diseases, National Institutes of Health, Centre d’Immunologie de Marseille-Luminy, Institut de Neurobiologie de la Méditerranée, Inserm, Centre National de la Recherche Scientifique, Aix-Marseille Université
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Variable analysis
independent variables
- Polyacrylamide gel type (4–12% NuPAGE or 10–20% Tris/glycine)
- Antibody concentration (1.66 µg/ml anti-PMY mAb and 1:3,000 human anti–ribosomal P antibody)
- Incubation time (overnight at 4°C)
- Protein transfer uniformity (assessed by Coomassie blue staining)
- Protein detection (by exposure to x-ray film after ECL substrate incubation)
- PVDF membrane material (Bio-Rad Laboratories)
- Blocking buffer (StartingBlock)
- Wash buffer (PBS and 0.1% Tween 20)
- ECL substrate (SuperSignal; Thermo Fisher Scientific)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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