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Antibody protocol for co-IPs (LI-COR antibodies)

SD Sabrina Dietz
MA Miguel Vasconcelos Almeida
EN Emily Nischwitz
JS Jan Schreier
NV Nikenza Viceconte
AF Albert Fradera-Sola
CR Christian Renz
AC Alejandro Ceron-Noriega
HU Helle D. Ulrich
DK Dennis Kappei
RK René F. Ketting
FB Falk Butter
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For co-IP experiments, we first probed the IP bait with HRP-linked secondary antibodies, as described above. Then, we probed for the co-IP using LI-COR secondary antibodies. After incubation with primary antibody, as described above, membranes were washed and incubated with secondary antibodies compatible with the LI-COR System (FLAG/GFP: Licor IRDye® 680RD Donkey anti-Mouse IgG (H+L), #926-68072; Actin: Licor IRDye® 800CW Donkey anti-Rabbit IgG (H+L), #926-32213; both 1:15,000 in Skim Milk solution) for 1 h at room temperature. After three additional washes with PBS-T, the membranes were imaged using an Odyssey CLx scanner and processed using Image Studio software (LI-COR, Version 3.1).

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