A DNA-Acrylamide Gel Column for Analyzing Proteins That Bind to DNA, I. DNA Polymerase

Abstract

Abstract. A new procedure is described for purifying proteins that specifically bind to DNA. DNA is entrapped in polyacrylamide gel particles which can then be used in standard column chromatographic procedures. The method was developed using Escherichia coli DNA polymerase as the test material. The crude enzyme was applied at low ionic strength and eluted at high ionic strength with a 200-fold increase in specific activity on a single passage through the column. The method is versatile and simple and is not restricted to DNAprotein systems. Any macromolecule can be entrapped in the gel particles; these can interact with other large or small molecules in the liquid phase. The gel is stable at elevated temperatures and can therefore be used in hybridization experiments.The binding of DNA-specific proteins to DNA provides the basis for a highly efficient and relatively simple procedure for isolating and purifying such proteins. Several methods have been described1-4 for immobilizing DNA on solid matrices which are then employed in standard column chromatographic procedures. We present here a new method in which the immobile matrix is composed of polyacrylamide gel particles. Aside from being versatile and simple, the method has the main advantages that no chemical steps are needed to fix the DNA in the matrix and the same column can be used repeatedly. The gel is made by dissolving DNA in the monomer solution before polymerization. The pore size of the gel particle is such that the DNA remains trapped, unless it is attacked enzymatically. So far, we have used only native DNA in the gel, but there is no reason why denatured DNA, RNA, or in fact any other macromolecule cannot be used since the pore size may be varied at will; therefore, even small molecules can be entrapped in the particles. The gel is stable at elevated temperatures, so that it is also possible to carry out hybridization experiments using this method. If the molecules that one seeks to purify are too large to enter the gel particle, this procedure can still be used, for there appears to be enough immobilized DNA protruding from the surface to be able to interact efficiently with the molecules in the liquid phase.We report here the details of this new procedure using as a test material a crude fraction of DNA polymerase previously described.5 One passage through the DNA-gel column increased the specific activity about 200 times. The physical characterization of this polymerase will be described in detail elsewhere.

Phosphorylation and DNA binding of nuclear rat liver proteins soluble at low ionic strength

“…Following two-dimensional gel electrophoresis, the gels were dried under vacuum and subjected to autoradiography (Olson et al, 1974b).3 DNA-Polyacrylamide Column Chromatography. DNApolyacrylamide column chromatography was performed as previously described (Cavalieri and Carroll, 1970;Vaughan and Comings, 1973). Rat liver DNA was prepared by a modified Marmur procedure (Marmur, 1961;Sitz et al, 1973).…”

DNA Affinity Chromatography of Transcription Factors

“…The stationary phase is composed of polyacrylamide gel particles containing entrapped DNA (57). The DNA-entrapped gel is prepared by combining the DNA with acrylamide and bis-acrylamide followed by addition of TEMED.…”

UV-Irradiated DNA Matrixes Selectively Bind Endocrine Disruptors with a Planar Structure

“…These properties make DNA alone difficult to utilize as a functional material. There are several reports on the utilization of modified DNA as a functional material, including DNA-columns ( , ), DNA/nanoparticle hybrid materials ( , ), DNA-films ( − ), DNA-gels ( − ), and DNA-immobilized nonwoven cellulose fabric (). DNA materials have been used for the isolation of DNA-binding proteins, molecular devices, and optical materials.…”