Abstract
Purpose: :
NRL is a key transcription factor that controls rod photoreceptor development and function. Mutations in the NRL gene lead to retinal disease. Microarray studies have revealed that a large number of photoreceptor genes are regulated by NRL. However, the mechanism of transcriptional regulation of these down-stream targets is still poorly understood. The goal of this study is isolate NRL-containing transcriptional complexes and identify regulatory proteins that are part of these complexes.
Methods: :
Bovine retinas were processed for protein purification. Nuclear fraction was obtained by differential centrifugation. Ammonium sulfate fractionation was performed, and NRL positive fractions were further enriched by Superdex-200 gel filtration column. Finally, high molecular mass fractions with NRL containing complexes were immunopurified using NRL-IgG coupled agarose. After SDS-PAGE of NRL-complex fraction, the proteins are being analyzed by mass spectrometry. The presence of NRL, CRX and other proteins was monitored by immunoblot analysis.
Results: :
Most of the NRL-containing complexes were enriched in 0-20% and 20-40% ammonium sulfate fractions. The high molecular mass fractions (>200 kDa) after gel filteration containing NRL were immunopurified. Immunoblot analysis of the resulting protein complexes showed the presence of CRX, further establishing the close synergistic interaction of NRL and CRX in controlling gene regulation in the retinal photoreceptors. Further analysis of the proteins in NRL-containing complexes is in progress by mass sspectrometry as well as immunoblot analysis using candidate antibodies.
Keywords: transcription factors • protein purification and characterization • proteomics