Oligonucleotide trapping method for transcription factor purification systematic optimization using electrophoretic mobility shift assay.

Abstract

Oligonucleotide trapping, where a transcription factor-DNA response element complex is formed in solution and then recovered (trapped) on a column, was optimized for the purification of CAAT/enhancer binding protein (C/EBP) from rat liver nuclear extract. Electrophoretic mobility shift assays (EMSAs) with ACEP24(GT)5 oligonucleotide, containing the CAAT element, was used to estimate thebinding affinity and concentration of C/EBP in the nuclear extract and then low concentrations of protein and oligonucleotide, which favor specific binding, were used for all further experiments. Also using EMSA, the highest concentrations of competitors, which inhibit non-specific binding but do not inhibit oligonucleotide binding by C/EBP, were determined to be 932 nM T18 (single-stranded DNA), 50 ng/ml heparin (non-DNA competitor), and 50 microg/ml poly(dI:dC) (duplex DNA). Inclusion of 0.1% Tween-20 improved DNA binding. For complex formation, 110 microg nuclear extract was diluted to 0.2 nM C/EBP (apparent Kd of C/EBP) and 1.34 nM ACEP24(GT)5 was added, along with Tween-20 and the competitors. After incubation, the complex was trapped by annealing the (GT)5 tail of the C/EBP-[ACEP24(GT)5] complex to an (AC)5-Sepharose column under flow at 4 degrees C. The column was washed with 0.4 M NaCl and the protein eluted with 1.2 M NaCl. The purification typically resulted in two proteins of apparent molecular mass 32000 and 38000. The smaller one, the major product, was identified to be C/EBP-alpha. The yield was 2.1 microg (66 pmol) of purified C/EBP-alpha p32. This systematic approach to oligonucleotide trapping is generally applicable for the purification of other transcription factors.