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. 1997 Feb 10;136(3):487–500. doi: 10.1083/jcb.136.3.487

Figure 4.

Figure 4

Purified Abp1p binds in vitro to c2-10 central core DNA. (A) Biochemical purification of the c2-10 binding activity was monitored by mobility shift assays (top) and silver-stained SDSPAGE (bottom). See Materials and Methods for details of the purification procedure. Mobility shift assays used 32P–c2-10 DNA as probe (first lane) and contained 20 μl of the diluted chromatin extract in binding cocktail applied to the affinity column (load; L), 20 μl of the column flowthrough (FT), 20 μl of the column wash fraction (W), or 2 μl of the 0.5-M KCl eluate fractions (lanes 1–6). Amounts shown on the 7.5% SDS-PAGE gel are 2 μl of the column load, 2 μl of the column flowthrough, 5 μl of the column wash, or 12.5 μl of the 0.5-M KCl eluate fractions. Sizes indicated for the molecular mass standard (first lane) are in kilodaltons. Affinity chromatography resulted in an ∼2,000fold purification of Abp1p compared with the specific activity found in low-salt extract from whole cells (see Table III). (B) Western Blot analysis of affinity-purified elution fractions using a polyclonal antibody to Abp1p. αAbp1p recognizes a 62-kD protein band in 0.4 M KCl extract from wild-type SBP120390 or overexpressing SBP120390/pSp200-abp1 cells as well as in eluate fractions 2 and 3 from the DNA affinity column (left), but it does not recognize a crossreacting protein in 0.4-M KCl extract prepared from an abp1 null (right). Size standards are not indicated for the right panel, but they migrated similarly to those shown for the left panel. (C) A supershift of fragment c2-10 is observed with αAbp1p and affinity-purified p62. Mobility shift assays were performed with 1.2 μg of protein in a 0.4-M KCl chromatin extract from wild-type SBP120390 or overexpressing SBP120390/pSp200-abp1 cells, or with 1 ng elution fraction 3 protein mixed with increasing amounts of polyclonal antibody to Abp1p (0.01 or 0.1 μl). αAbp1, in the absence of S. pombe extract, has no effect. (D) A 0.4-M KCl chromatin extract prepared from an abp1 null strain lacks c2-10 DNA-binding activity.