Purification Of DNA-binding Proteins Research Articles

Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

Determination of BRCA2 oncosuppressor protein expression in human mammary cells by affinity perfusion high-performance chromatography

In this work, we report a method for the determination of BRCA2 oncosuppressor protein in human mammary cells by affinity perfusion chromatography. This method involves labeling proteins with [ 35S]-methionine. The isolation and purification of DNA-binding proteins was performed by affinity chromatography on Heparin POROS 20HE. BRCA2 proteins, known to act in the transcriptional control and in DNA repair activity, are included in the DNA-binding proteins. Specific immunoprecipitation was performed with anti-BRCA2 antibodies, and the immune complex [ 35S-BRCA2 proteins/anti-BRCA2 antibodies] was isolated by affinity chromatography on a Protein A POROS column. This procedure allows the determination of the percentage of BRCA2 among the DNA-binding proteins and the quantitation of the difference of expression of BRCA2 oncosuppressor protein in breast carcinoma cells and in normal breast cells treated or untreated with phytoestrogens, such as daidzein or genistein.

Solid-phase technology: magnetic beads to improve nucleic acid detection and analysis

Magnetic separation of DNA, RNA or proteins has proven to be very useful for many applications in molecular biology. This chapter describes procedures used for mRNA isolation, subtractive hybridization, selective hybridization, sequencing, cloning, in vitro mutagenesis, detection and quantification of amplified material, sample preparation and purification of DNA-binding proteins. Solid-phase DNA sequencing and mRNA purification have been used most frequently. Among these are gene assembly strategies; new means to recover DNA binding fusion proteins and several magnetic bead based mutation assays. In addition, there are procedures for recovery of single-stranded DNA, labelling of single-stranded DNA probes, direct purification of tRNAs and reversible immobilization of antibodies. In many applications, magnetic bead technology provides simple, rapid, and reliable alternatives to traditional techniques. Both manual and automated methods can be developed and some of these will probably have great impact on molecular biology in the near future.

A new fast and easy method for the oligonucleotide affinity purification of DNA binding proteins

A new fast and easy method for the oligonucleotide affinity purification of DNA binding proteins

Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems: DNA-Protein Interactions.

Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems: DNA-Protein Interactions.

A new method for the preparation of DNA-cellulose

DNA immobilized on cellulose or agarose has been found very useful for the separation and purification of DNA-binding proteins (1–4). A variety of methods for DNA immobilization has been proposed, including absorption (3), entrapment in gels (4), and covalent binding (1). In the course of a study of the behavior of a complex between histones and immobilized DNA, we have met the need of a particularly stable DNA-cellulose preparation, i.e., involving not only covalent bonds between the nucleic acid and the polysaccharide, but bonds resistent to extremes of pH, particularly in the alkaline region. It has been found that cellulose activated with cyanuric chloride affords a convenient material for the preparation of DNA-cellulose, which has the following favorable characteristics: (i) It can be prepared by an easy and reproducible method; (ii) double-stranded DNA is bound in satisfactory amounts; (iii) the material is very stable, even in urea and in alkaline conditions.