Biotin 3 end dna labeling kit

We prepared nuclear extract from HuTu 80 cells with the commercially available NE-PER Nuclear and Cytoplasmic extraction kit from Thermo Scientific Pierce, Rockford, IL following the manufacturer’s protocol. For EMSA, a 28-bp minimal promoter region (a sequence: 5′-CCTCCGGGCAGAGCCCGCCCCAGCCACT −3′), a sequence between −60 and −33 was used. The promoter was biotinylated using a Biotin 3′-end DNA labeling kit (Thermo Scientific Pierce). LightShift Chemiluminescent EMSA kit from Thermo Scientific Pierce was used for EMSA. Briefly, for binding reactions the reaction volume was 20 μl with 5 μg of nuclear extract, 20 fmol of biotin end-labeled DNA, and 50 ng/μl poly (dI.dC), and was incubated at room temperature for 20–30 min. Analysis of oligonucleotide competition was carried out with a 50-fold molar excess of unlabeled SLC52A1 DNA fragment. For super-shift assays pretreatment of the nuclear extract was carried out with 2 μg anti-Sp-1 antibody or 2 μg normal rabbit IgG. Subsequently DNA-protein complexes were separated on a 6% DNA retardation gel (Invitrogen Life Technologies) in 0.5× Tris borate-EDTA buffer at 100V, followed by electrophoretic transfer of binding reactions to nylon membrane and crosslinking of transferred DNA to membrane in UV. Biotinilated DNA was detected by chemiluminescence using Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific Pierce).