Magnetic DNA affinity purification of ecdysteroid receptor.
A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.
Ozyhar
A
A
Ruhr-Universität Bochum, Lehrstuhl für Biochemie, Bochum, Germany.
Gries
M
M
Kiltz
H H
HH
Pongs
O
O
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D007447:Invertebrate Hormones / Q000302:isolation & purification*; D008722:Methods; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D011987:Receptors, Steroid / Q000302:isolation & purification*
D007447:Invertebrate Hormones; D009841:Oligonucleotides; D011987:Receptors, Steroid; C034808:ecdysteroid receptor; D004247:DNA