ChIP-MS
1) Protein complexes were cross-linked to the DNA using 1% formaldehyde before yeast chromatin fraction was prepared (Chymkowitch et al. Proc Natl Acad Sci U S A. 2012 Jun 26;109(26):10450-5)
2) 800 μg (Bradford assay) of this chromatin was used per Flag IP (anti-FLAG M2 magnetic beads, Sigma)
3) Bound complexes were washed once with ice-cold TBS + 0.01% NP40 + PIC.
4) Bound complexes were washed twice with ice-cold TBS + 350 mM NaCl + 0.01% NP40 + PIC.
5) Bound complexes were washed once with ice-cold TBS + 0.01% NP40 + PIC.
6) Bound complexes were eluted in TBS + 0.01% NP40 + 200 ng/μl 3XFLAG peptide (Sigma) + PIC, 1,5 hours at 25°C.
7) Beads were eliminated and the crosslink was reversed 15 min at 95°C.
8) Laemmli buffer was added before further incubation at 95°C during 5 min.
9) A TGX gel was ran (any KDa from Biorad) and stained using coomassie blue.
Lanes:
1-Molecular Mass ladder (Biorad Precision plus dual Xtra, 250 to 5 KDa)
2-IP:FLAG in control strain (no Flag-SUMO)
3-IP:FLAG in the FLAG-SUMO strain
4- IP:FLAG in the FLAG-SUMO strain treated with rapamycin
2) 800 μg (Bradford assay) of this chromatin was used per Flag IP (anti-FLAG M2 magnetic beads, Sigma)
3) Bound complexes were washed once with ice-cold TBS + 0.01% NP40 + PIC.
4) Bound complexes were washed twice with ice-cold TBS + 350 mM NaCl + 0.01% NP40 + PIC.
5) Bound complexes were washed once with ice-cold TBS + 0.01% NP40 + PIC.
6) Bound complexes were eluted in TBS + 0.01% NP40 + 200 ng/μl 3XFLAG peptide (Sigma) + PIC, 1,5 hours at 25°C.
7) Beads were eliminated and the crosslink was reversed 15 min at 95°C.
8) Laemmli buffer was added before further incubation at 95°C during 5 min.
9) A TGX gel was ran (any KDa from Biorad) and stained using coomassie blue.
Lanes:
1-Molecular Mass ladder (Biorad Precision plus dual Xtra, 250 to 5 KDa)
2-IP:FLAG in control strain (no Flag-SUMO)
3-IP:FLAG in the FLAG-SUMO strain
4- IP:FLAG in the FLAG-SUMO strain treated with rapamycin