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. 2002 Jun 1;30(11):2340–2348. doi: 10.1093/nar/30.11.2340

Figure 1.

Figure 1

Outline and optimisation of p53 DNA-binding assay. (A) Outline of experimental procedure. (B) Consensus (CON) or mutated (MUT) oligonucleotides were immobilised on beads and indicated amounts were incubated with 10 µg nuclear extract from WS-1 cells harvested 24 h after 35 J m–2 UVC irradiation. (C) Nuclear extract (20 µg) from UV-treated WS-1 cells was subjected to three consecutive binding reactions to CON oligonucleotide. As a negative control, a binding reaction with the MUT oligo is shown. The ratio of bound versus unbound amount of p53 is shown below the corresponding lanes. Oligonucleotides used for the binding were released from the beads as described in Materials and Methods and were run on an agarose gel containing ethidium bromide (Released DNA). (D) Immobilised CON oligonucleotide was used in a binding reaction with either 2.5 µg nuclear extract from UV-treated WS-1 cells (EXTRACT) or 600 ng purified, bacterially expressed p53 core domain (amino acids 92–312, p53 CORE). Nuclear extract from control cells was included to show the basal p53 levels. IN, input; B, bound fraction; UB, unbound fraction.