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Co-immunoprecipitation (co-IP) and Western blot

VI Valentina V. Ignatova
PJ Pascal W. T. C. Jansen
MB Marijke P. Baltissen
MV Michiel Vermeulen
RS Robert Schneider
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Whole cell extracts were prepared as described above. Total protein concentration was measured with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). 4 mg of whole cell extract was used per immunoprecipitation (IP). IP with GFP nanobody sepharose beads (Chromotek) was performed as described above. For Calnexin-IP, 10 μg of anti-Calnexin antibody from Abcam per IP was used. Input and IP samples were separated by SDS–PAGE and transferred to nitrocellulose membrane, followed by immunostaining. The antibodies used for immunostaining: anti-GFP (1:50; HMGU monoclonal antibody facility) or anti-Calnexin (1:1000; ab22595, Abcam) primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies goat anti-mouse (1:1500, P0447, Dako) and anti-rabbit (1:100000, 111-035-003, Jackson Laboratory) correspondingly.

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