Journal:

Article Title: An Ikaros-Containing Chromatin-Remodeling Complex in Adult-Type Erythroid Cells

doi:

Figure Lengend Snippet: PYR complex DNA-binding activity cofractionates from MEL nuclear extract with Ikaros and a subpopulation of SWI/SNF and NuRD protein. (A) Purification scheme. The PYR complex was purified from MEL nuclear extract in four chromatographic steps using High Q Macroprep (Bio-Rad; 0.1 to 0.4 M NaCl step elution), reactive yellow 3-agarose (Sigma; 0.1 to 1.0 M NaCl gradient), double-stranded (ds) DNA-cellulose (calf thymus DNA-cellulose [Sigma], 0.1 to 0.7 M NaCl step gradient), and Superose 6 (Amersham Pharmacia) columns. (B) Chromatography on reactive yellow 3-agarose shows peak PYR complex DNA-binding activity by gel shift assay (PYR) eluting in fractions 64 through 70 (upper panel), corresponding precisely with the elution pattern of Ikaros by Western blotting (lower panel). The majority of NuRD (Mi-2) and SWI/SNF (BRG1, SRG3, and BAF57) proteins elute prior to fraction 64, with some overlap into fractions 64 through 68. Fractions 65 through 70 were pooled, concentrated, adjusted to 0.1 M NaCl, and loaded onto the DNA-cellulose column (C). Peak PYR complex DNA-binding activity elutes off the DNA cellulose column in fractions 0.3 through 0.45 (upper panel), again corresponding precisely with the elution pattern of Ikaros (lower panel). Much of the NuRD (Mi-2 and RbAp46/48) and SWI/SNF (BRG1, SRG3, and BAF57) proteins elutes prior to fraction 0.3, but there is greater overlap into the PYR DNA-binding fractions than seen with reactive yellow 3-agarose. Fractions 0.3 through 0.45 were pooled, concentrated, and loaded onto the Superose 6 gel filtration column (D). Again, PYR complex DNA-binding activity, which elutes in high-molecular-mass fractions (1 to 2 MDa, upper panel), corresponds exactly with the elution pattern of Ikaros by Western blotting (lower panel). Here, however, both NuRD (Mi-2 and RbAp46/48) and SWI/SNF (BRG1 and BAF57) subunits cofractionate very closely with PYR DNA-binding activity and Ikaros. FT, failure to bind.

Article Snippet: The PYR complex was purified from MEL nuclear extract in four chromatographic steps using High Q Macroprep (Bio-Rad; 0.1 to 0.4 M NaCl step elution), reactive yellow 3-agarose (Sigma; 0.1 to 1.0 M NaCl gradient), double-stranded (ds) DNA-cellulose (calf thymus DNA-cellulose [Sigma], 0.1 to 0.7 M NaCl step gradient), and Superose 6 (Amersham Pharmacia) columns. (B) Chromatography on reactive yellow 3-agarose shows peak PYR complex DNA-binding activity by gel shift assay (PYR) eluting in fractions 64 through 70 (upper panel), corresponding precisely with the elution pattern of Ikaros by Western blotting (lower panel).

Techniques: Binding Assay, Activity Assay, Purification, Chromatography, Electrophoretic Mobility Shift Assay, Western Blot, Filtration

Journal:

Article Title: An Ikaros-Containing Chromatin-Remodeling Complex in Adult-Type Erythroid Cells

doi:

Figure Lengend Snippet: Ikaros, NuRD, and SWI/SNF proteins coimmunopurify from chromatography fractions containing peak PYR complex DNA-binding activity. (A) Ikaros and Mi-2 coimmunoprecipitate from reactive yellow 3-agarose-fractionated MEL extract. (Left panel) Western blot analysis with rabbit polyclonal anti-Ikaros antibody. Mouse monoclonal antibodies used for immunoprecipitation (IP) are indicated above lanes 2 through 4. The sample for lane 1 was 1/10 the volume of reactive yellow 3-agarose-purified PYR complex (yellow 3 fraction 67 [Y3]) before precipitation as a positive control for the Ikaros signal (Ik-1, 65 kDa; Ik-2, 55 kDa). Weak cross-reaction of the anti-rabbit IgG secondary antibody with the mouse IgG heavy chain results in a faint band of approximately 50 kDa in lanes 2 through 4. (Right panel) Western blot analysis with an anti-Mi-2 rabbit polyclonal antibody. Lane 1, reactive yellow 3-agarose fraction 67; lane 2, anti-Aiolos precipitate; lane 3, anti-Mi-2 precipitate; lane 4, anti-Ikaros precipitate. (B) Ikaros, BRG1, and Mi-2 copurify with BAF57 off an anti-BAF57 immunoaffinity column. The start material (Start) was DNA-cellulose-purified PYR complex. The vast bulk of the protein loaded onto the column does not bind (FT). This was confirmed by silver staining (data not shown). The column was washed twice with 0.1 M NaCl buffer (0.1 A and B) and twice with 0.5 M NaCl buffer (0.5 A and B) and then eluted with 0.1 M glycine (pH 2.5) (Elute). BAF57 and Ikaros, particularly Ikaros isoform 2, bind very tightly to the column, withstanding the 0.5 M salt washes. Both BRG1 and Mi-2 bind to the column with somewhat lower affinity, eluting in the 0.5 M salt and pH 2.5 glycine fractions. (C) Ikaros, NuRD, and SWI/SNF proteins copurify with Mi-2 off an anti-Mi-2 immunoaffinity column. The start material (Start) was DNA-cellulose-purified PYR complex. Silver-stained gels showed that the vast bulk of the protein loaded onto the column does not bind (data not shown). The column was washed and eluted as described for panel B. Mi-2 binds very tightly to the column, with much of the Mi-2 remaining on the column after the pH 2.5 elution step. HDAC2, Ikaros isoforms 1 and 2, and SWI/SNF proteins (BAF57, BRG1, and BAF155) bind to the column with high affinity, eluting only in the pH 2.5 glycine fraction.

Article Snippet: The PYR complex was purified from MEL nuclear extract in four chromatographic steps using High Q Macroprep (Bio-Rad; 0.1 to 0.4 M NaCl step elution), reactive yellow 3-agarose (Sigma; 0.1 to 1.0 M NaCl gradient), double-stranded (ds) DNA-cellulose (calf thymus DNA-cellulose [Sigma], 0.1 to 0.7 M NaCl step gradient), and Superose 6 (Amersham Pharmacia) columns. (B) Chromatography on reactive yellow 3-agarose shows peak PYR complex DNA-binding activity by gel shift assay (PYR) eluting in fractions 64 through 70 (upper panel), corresponding precisely with the elution pattern of Ikaros by Western blotting (lower panel).

Techniques: Chromatography, Binding Assay, Activity Assay, Western Blot, Immunoprecipitation, Purification, Positive Control, Silver Staining, Staining