Journal: Journal of Biological Chemistry

Article Title: Human Ku Antigen Tightly Binds and Stabilizes a Tetrahelical Form of the Fragile X Syndrome d(CGG) Expanded Sequence

doi: 10.1074/jbc.m005542200

Figure Lengend Snippet: FIG. 1. Resolution of d(CGG)8 binding activities by d(CGG)16- Sepharose affinity chromatography and SDS-PAGE of protein fractions. Phosphocellulose-purified fraction of the d(CGG)8 binding protein was loaded onto a d(CGG)16-Sepharose affinity column, and proteins were eluted by a stepwise gradient of 0.05–1.0 M NaCl in buffer D. Each eluting salt solution (a single column volume) was collected in two fractions that were stabilized by 0.2 mg/ml STI and 0.05% Nonidet P-40. A, mobility shift electrophoresis of the affinity-purified protein. Fractions were assayed for binding of 32P-59-labeled d(CGG)8 as de- scribed under “Experimental Procedures.” Concentrations of eluting salt are indicated for each fraction in the abscissa. B, Coomassie Blue staining of SDS-PAGE resolved affinity purified protein fractions. Elec- trophoresis and protein staining were conducted as described under “Experimental Procedures.” To better separate high molecular mass proteins, lower mass proteins including the added STI stabilizer were run out of the gel. Arrows indicate the positions of the 72- and 87-kDa protein bands whose distribution in the eluted fractions was in concord- ance to the binding activity (A).

Article Snippet: Materials and Enzymes—Isotopically 59-labeled [g-32P]ATP (;3000 Ci/mmol), Bacteriophage T4 polynucleotide kinase, and molecular mass Rainbow® marker proteins were the products of Amersham Pharmacia Biotech.

Techniques: Binding Assay, Affinity Chromatography, SDS Page, Purification, Affinity Column, Electrophoretic Mobility Shift Assay, Affinity Purification, Labeling, Staining, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Human Ku Antigen Tightly Binds and Stabilizes a Tetrahelical Form of the Fragile X Syndrome d(CGG) Expanded Sequence

doi: 10.1074/jbc.m005542200

Figure Lengend Snippet: FIG. 2. Covalent cross-linking of the binding protein to d(CGG)8. Resolution of the d(CGG)8 binding activity by d(CGG)16- Sepharose affinity column chromatography was conducted as described in the legend to Fig. 1. Aliquots of each of the d(CGG)16-Sepharose resolved fractions were incubated with 32P-59-labeled d(CGG)8 under binding conditions as described under “Experimental Procedures” ex- cept that a 100-fold molar excess of unlabeled ;d(G)17; competing oligomer was present in the reaction mixtures. The protein-d(CGG)8 complexes were either directly resolved by nondenaturing mobility shift electrophoresis or were covalently cross-linked by UV light (see “Exper- imental Procedures”). Following electrophoresis through an SDS-12% polyacrylamide gel, the dried gels were exposed to autoradiographic film. Molecular mass of the cross-linked complex was estimated from its migration relative to that of molecular size marker proteins. A, nonde- naturing mobility shift electrophoresis. B, SDS-PAGE of a UV-cross- linked complex.

Article Snippet: Materials and Enzymes—Isotopically 59-labeled [g-32P]ATP (;3000 Ci/mmol), Bacteriophage T4 polynucleotide kinase, and molecular mass Rainbow® marker proteins were the products of Amersham Pharmacia Biotech.

Techniques: Binding Assay, Activity Assay, Affinity Column, Chromatography, Incubation, Labeling, Electrophoretic Mobility Shift Assay, Electrophoresis, Migration, Marker, SDS Page