Western blotting procedures were previously described [12 (link), 13 (link)]. Briefly, protein samples were separated by SDS-PAGE on a 10% gel, soaked in transfer buffer, and transferred to a PVDF membrane. The membrane was incubated with primary Abs overnight then with HRP-conjugated secondary Abs for 60 min at room temperature. Protein bands were visualized with an ECL Prime kit (GE Healthcare UK, Ltd.), and immunoblotting signals were acquired with a GENEGNOME Bio Imaging System (SYNGENE).
Okada M., Kawagoe Y., Sato Y., Nozumi M., Ishikawa Y., Tamada A., Yamazaki H., Sekino Y., Kanemura Y., Shinmyo Y., Kawasaki H., Kaneko N., Sawamoto K., Fujii Y, & Igarashi M. (2021). Phosphorylation of GAP-43 T172 is a molecular marker of growing axons in a wide range of mammals including primates. Molecular Brain, 14, 66.
Other organizations :
Niigata University, Gunma University, The University of Tokyo, Osaka National Hospital, Kanazawa University, Nagoya City University, Niigata University Medical and Dental Hospital
Incubation time of secondary Abs (60 min at room temperature)
Transfer buffer
PVDF membrane
10% SDS-PAGE gel
ECL Prime kit (GE Healthcare UK, Ltd.)
GENEGNOME Bio Imaging System (SYNGENE)
controls
Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.
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