Method for trapping affinity chromatography of transcription factors using aldehyde–hydrazide coupling to agarose

Abstract

The uses of a method of coupling DNA is investigated for trapping and purifying transcription factors. Using the GFP-C/EBP fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry utilized is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA-binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose which couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes including E2A, c-myc, and myo-D were also purified but myogenenin and NFκB were not. Therfore, this approach proved valuable for both affinity chromatography and for the trapping approach.

Topochemical Engineering of Cellulose-Based Functional Materials

“… 97 − 101 This was similar to a coupling reaction between an aldehyde and a primary amine, which led to the formation of Schiff’s base in a weakly acidic environment. 101 In our case, a stable hydrazone was formed as supported by the literature, 102 − 104 thus beads were made into zwitterions in nature.…”

“…In a consecutive oxidation step, −CHO got converted to −COOH by NaClO 2 , generating NaClO, which then reoxidized hydroxylamine to the N -oxoammonium ion. , The carbonyl groups (−CHO) formed during the oxidation process were further coupled with hydrazides containing quaternary ammonium ions through the click chemistry approach. − This was similar to a coupling reaction between an aldehyde and a primary amine, which led to the formation of Schiff’s base in a weakly acidic environment . In our case, a stable hydrazone was formed as supported by the literature, − thus beads were made into zwitterions in nature.…”

Protein–DNA/RNA Interactions: An Overview of Investigation Methods in the -Omics Era

“…Several procedures have been explored to bind the oligonucleotide bait to the polymeric support, with two methods being the most successful. The nucleotide sequence can be covalently bound by chemical cross-links on a suitably derivatized resin; alternatively, the oligonucleotide bait can be labeled with biotin and immobilized on streptavidin-coated beads. − Binding buffer composition also exerts a critical role in favoring specific DNA–protein interactions. Physicochemical characteristics including pH value, the concentration of mono- and bivalent ions, the addition of specific DNA competitors to reduce nonspecific binding (poly dI, AD, etc.…”