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. 1999 Mar 30;96(7):3595–3599. doi: 10.1073/pnas.96.7.3595

Figure 4.

Figure 4

Analysis of hetero-oligomerization between p53tet mutants and wild-type human p53tet. The second procedure described in Materials and Methods was followed. A total of 2 nmol of His-tagged tetL were incubated either alone (lane 1) or with nontagged wild-type tetS (lane 2), mutant L332V/F341L/L344I (lane 3), or L332V/M340I/F341L (lane 4). The His-tagged homo-oligomers and the hetero-oligomers formed were separated from the remaining nontagged homo-oligomers by specific binding to Ni-NTA beads followed by elution with imidazole. Lanes 5–14, eluates containing His-tagged homo-oligomers and hetero-oligomers (if applicable) from tetL alone (lane 5) or mixtures of tetL with wild-type tetS (lanes 6–8), L332V/F341L/L344I (lanes 9–11), or L332V/M340I/F341L (lanes 12–14). TetL:tetS (wild-type or mutants) molar ratios in the corresponding incubation mixtures were 1:0.5 (lanes 6, 9, and 12), 1:1 (lanes 7, 10, and 13), or 1:2 (lanes 2–4, 8, 11, and 14). The conditions for the experiment were set up by using the alternative procedure described in Materials and Methods. In control samples with individual proteins, no His-tagged tetL was found in the washing fractions, and no untagged wild-type or mutant tet were found in the fractions eluted. Similar results consistent with those shown here were obtained in repeated experiments.