Proteins were extracted and separated as described previously (Baier et al., 2018 (link)) using 10% SDS‐Polyacrylamide gels, followed by colloidal Coomassie staining (Dyballa and Metzger, 2009 (link)) and Western blotting onto a nitrocellulose membrane (AmershamTM; GE Healthcare, Uppsala, Sweden). After blocking with 2.5% (w/v) BSA and 2.5% (w/v) milk powder in TBS‐T buffer, immunodetection was carried out via incubation with horseradish peroxidase coupled Strep II tag (IBA Life Sciences, Göttingen, Germany) or HA‐tag antibodies (Thermo Scientific) in blocking buffer (1:10000), washing with TBS‐T and addition of Pierce™ ECL Western blotting substrates (Thermo Scientific), followed by detection in a Fusion Fx7 chemiluminescence imaging system (Vilber Lourmat).
Protocol detail
Protein Extraction and Analysis Protocol
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Antibodies
Buffer
Chemiluminescence
Horseradish peroxidase
Membrane
Milk
Nitrocellulose
Polyacrylamide gels
Powder
Proteins
Strep tag
Corresponding Organization : Bielefeld University
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Variable analysis
independent variables
- Protein extraction and separation method (10% SDS-Polyacrylamide gels)
- Protein detection by colloidal Coomassie staining
- Protein detection by Western blotting
- Blocking with 2.5% (w/v) BSA and 2.5% (w/v) milk powder in TBS-T buffer
- Incubation with horseradish peroxidase coupled Strep II tag or HA-tag antibodies (1:10000) in blocking buffer
- Washing with TBS-T
- Addition of Pierce™ ECL Western blotting substrates
- Detection in a Fusion Fx7 chemiluminescence imaging system
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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