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Article Title: ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly

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Figure Lengend Snippet: Purification of the native form of Acf1 leads to the isolation of ACF comprising Acf1 (p170 and p185) and ISWI. (A) Scheme for the purification of native form of Acf1 from Drosophila embryos. (B) Hydroxyapatite chromatography. The peak gradient fractions from the Source 15Q (Pharmacia Biotech) column were applied to a Bio-Gel HT hydroxyapatite (Bio-Rad) column, and protein was eluted with a linear potassium phosphate gradient. The column fractions were subjected to Western blot analysis with antibodies against Drosophila Acf1 (p170/p185), ISWI, topoisomerase II, and dCAF-1 p55 in conjunction with 125I-labeled protein A. With the Acf1 Western blot, the p170 and p185 forms of Acf1 were not clearly resolved. Also, the slower migrating species that cross-reacts with the Acf1 antiserum is not recognized by the affinity-purified antibodies (e.g., see Fig. ​Fig.2).2). (C) POROS heparin chromatography. The peak hydroxyapatite fractions were applied to a POROS heparin (PerSeptive Biosystems) column, and protein was eluted with a linear NaCl gradient. The column fractions were subjected to Western blot analysis, as in B. The control sample is an ACF-containing fraction from the Source 15Q chromatography step. The p170 and p185 forms of Acf1 were not clearly resolved. (D) Glycerol gradient sedimentation. The peak POROS heparin fractions were subjected to 15%–40% (vol/vol) glycerol gradient sedimentation. The glycerol gradient fractions were subjected to Western blot analysis, as in B and C. The p170 and p185 forms of Acf1 were not clearly resolved. (E) Micrococcal nuclease digestion analysis. ACF activity in the glycerol gradient fractions was tested by micrococcal nuclease digestion analysis. Chromatin assembly reactions contained 10 μl of each 400 μl fraction and were carried out as described in Materials and Methods. The samples were then partially digested with two different concentrations of micrococcal nuclease. The resulting DNA fragments were deproteinized, resolved by 1.5% agarose gel electrophoresis, and visualized by staining with ethidium bromide. The mass markers (M) are the 123-bp DNA ladder (GIBCO-BRL). The peak of ACF activity is seen in fractions 7–9. (F) Native ACF consists of Acf1 (p185 and p170) and ISWI. Glycerol gradient fractions were subjected to 6% polyacrylamide–SDS gel electrophoresis, and proteins were visualized by silver staining. The sizes of molecular mass markers and the ACF subunits are indicated. The traces of dCAF-1 p55/NURF-55 that were seen in Western blots of the glycerol gradient fractions (D) could not be detected in these silver-stained SDS–polyacrylamide gels.

Article Snippet: The dialyzed Bio-Gel HT hydroxyapatite fractions were applied directly to a POROS 20 HE1 heparin (PerSeptive Biosystems) resin [column volume = 0.2 ml, column dimensions (diam.

Techniques: Purification, Isolation, Chromatography, Western Blot, Labeling, Affinity Purification, Sedimentation, Activity Assay, Agarose Gel Electrophoresis, Staining, SDS-Gel, Electrophoresis, Silver Staining