Hybond n nucleic acid transfer membrane

Genomic DNA digestion was achieved by using DNA (1000 ng) in a total volume of 100 μl overnight digestion, with an appropriate restriction enzyme. Following an agarose gel electrophoresis of the digested genomic DNA samples, the DNA was transferred to a Hybond N+ nucleic acid transfer membrane (Amersham Biosciences) through capillary action. A DIG High Prime DNA Labelling and Detection Kit (Roche) was used to generate Digoxigenin (DIG) – labelled DNA probe. Hybridization of the membrane as well as detection were performed according to the kit manufacturer’s instructions. The membrane was viewed under a Fusion Chemiluminescence Camera (Fisher Biotech).

The pET28a vector carrying the full-length HY5 cDNA fragment (Hajdu et al., 2018 (link)) was introduced into Escherichia coli BL21 DE3 Rosetta cells (New England 513 Biolabs). Proteins were purified on His-Select Nickel affinity gel (SIGMA). 2 µg of purified protein was incubated for 30 min with 2 pmol biotin-labeled DNA (respective P5CS1 and PDH1 oligonucleotide sequences are available in ). DNA fragments and complexes were separated in 4% (W/V) native polyacrylamide gel, then blotted to HyBond-N⁺ nucleic acid transfer membrane (Amersham). DNA fragments were crosslinked to the membrane with UV light (UV Stratalinker, Stratagene). DNA fragments were detected by an immune reaction with Streptavidin-conjugated horseradish-peroxidase (Thermo Scientific) using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific). Signals were developed with a chemiluminescent substrate (Supersignal West-Thermo Scientific) and detected in Fusion FX western blot and gel documentation imaging device (Vilber). Experiments were repeated twice.