Increasing the activity of affinity-purified DNA-binding proteins by adding high concentrations of nonspecific proteins.
A large decrease in the activity of two sequence-specific DNA-binding proteins implicated in transcription control was seen when these were affinity purified and assayed under standard conditions in electrophoretic mobility shift assays. Increasing the concentration of bovine serum albumin in the reaction mixtures from 0.1 to 5 mg/ml stimulated the DNA-binding activity of these affinity-purified proteins, human CREB (cyclic AMP response element binding protein) and MDBP (methylated DNA-binding protein), approximately 5-to more than 20-fold. In the case of affinity-purified MDBP, adding back the affinity flow-through fraction to the assay mixture gave similar extents of stimulation at much lower final protein concentrations. The specific DNA-binding activity of the affinity-purified CREB, but not that of MDBP, was also increased by adding a nonionic detergent to the binding reaction buffer although not as much. The large increase in the amount of MDBP.DNA complex seen upon supplementation of the affinity-purified MDBP with the affinity flow-through fraction or 5 mg/ml of BSA was shown to be due to stimulation, by nonspecific proteins, of specific complex formation and not to prevention of activity losses by adsorption or denaturation during the assay.
Zhang
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XY
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112.
Asiedu
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CK
Supakar
P C
PC
Ehrlich
M
M
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D017362:Cyclic AMP Response Element-Binding Protein; D004247:DNA; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D012710:Serum Albumin, Bovine
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D012710:Serum Albumin, Bovine; D004247:DNA