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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