PCNA Antibody (PC10): sc-56

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:5000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:5000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

To generate SUMOylated StrepII-PCNA for Fig. 4b, in vitro SUMOylation was carried out at 37 °C for 4 h using a SUMOylation kit (Enzo). SUMOylation reactions contained 1 mg StrepII-PCNA and 1 mg His-SUMO2. Upon completion, SUMOylation reactions were diluted with 10 volumes of Ni-NTA binding buffer (50 mM Tris pH 8.0, 0.3 M NaCl, 10% glycerol, 0.1% Triton-X-100, 5 mM imidazole) and incubated with Ni-NTA beads overnight at 4 °C. After washing 10× with 50 volumes of binding buffer, the His-SUMO2-conjugated StrepII-PCNA proteins were eluted with PBS containing 500 mM imidazole. Eluted fractions were diluted with 10 volumes of PBS and incubated with Strep-Tactin beads for 2 h at 4 °C, followed by 10× washes with PBS containing 0.1% Triton-X-100. For protein-protein interactions shown in Fig. 4b, 200 µl of the CB fraction was added to Strep-Tactin beads bound with the indicated Strep-tagged proteins and incubated for 4 h at 4 °C. Unbound proteins were removed by extensive wash with FLAG-binding buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 250 mM NaCl, 0.1% Triton-X-100, 10% Glycerol), and bound proteins were analyzed by western blots. Similar pull-down was performed to generate data presented in Fig. 4e, except that the CB fractions were prepared from HEK293T cells with or without FLAG-SSRP1 expression and Strep-tagged S2-PCNA fusion was used. For Fig. 4c, e, FLAG-CAF1A was first purified from either HEK293T cells (Fig. 4c) or E. coli (Fig. 4d) and bound to FLAG M2 agarose beads (Sigma). The FLAG-CAF1A-containing beads were then incubated with either StrepII-PCNA or StrepII-S2-PCNA purified from E. coli. The unbound proteins were removed and the bound proteins were analyzed, as described above.

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