Forty
whole brains without cerebellums from wild-type (negative control)
or BAC transgenic mice (ages from P14 to P17) were used for affinity
purification. The P2 fraction was cross-linked by DSP, and the membrane-bound
proteins were extracted as described previously. The cleared extraction
was incubated with monoclonal anti-FLAG-M2 magnetic beads (100 μL,
Sigma) at 4 °C overnight on a rotator. Anti-FLAG beads were pelleted
by a magnetic rack and washed five times with TBS containing 1% Triton
X-100. The bound proteins were eluted with 3×FLAG peptide according
to the manufacturer’s instruction (Sigma). The purified complexes
were resolved on a SDS-PAGE gel. 10% of the complexes were used for
immunoblot analysis, and 90% were used for SYPRO Ruby staining (Bio-Rad).
Protein bands were visualized using a UV light, and the image was
taken using an LAS-1000plus system (Fujifilm). The whole lane was
excised into 21 fractions and stored at −80 °C for further
mass spectrometry analysis.
whole brains without cerebellums from wild-type (negative control)
or BAC transgenic mice (ages from P14 to P17) were used for affinity
purification. The P2 fraction was cross-linked by DSP, and the membrane-bound
proteins were extracted as described previously. The cleared extraction
was incubated with monoclonal anti-FLAG-M2 magnetic beads (100 μL,
Sigma) at 4 °C overnight on a rotator. Anti-FLAG beads were pelleted
by a magnetic rack and washed five times with TBS containing 1% Triton
X-100. The bound proteins were eluted with 3×FLAG peptide according
to the manufacturer’s instruction (Sigma). The purified complexes
were resolved on a SDS-PAGE gel. 10% of the complexes were used for
immunoblot analysis, and 90% were used for SYPRO Ruby staining (Bio-Rad).
Protein bands were visualized using a UV light, and the image was
taken using an LAS-1000plus system (Fujifilm). The whole lane was
excised into 21 fractions and stored at −80 °C for further
mass spectrometry analysis.