Exemplary electrophoretic mobility agarose gel shift assay
The promoter fragments (100–200 bp) harboring central MatInspector predicted (bipartite) DR3-type VDREs for CRABP2 (forward: 5′-CAAGCCCCTCTCTGAGTACG-3′, reverse: 5′-GATGCTCAGGGCTCGTGTAT-3′), OLFML3 (5′-TCCCAGTGGTAAGGAAGTCAG-3′, 5′-TTGCTTGAAAGAGGCCAGAT-3′), BNC2 (5′-GTCAAGCATGCCTGTGAAGA-3′, 5′-GGCCCTCCTTATTTCAGTCC-3′), TMEM91 (5′-CCACGATCACTTCACTGCAC-3′, 5′-GCCTTAACCAAGGACCTTCC-3′), and KRT19 (5′-CTGCTGTGAGGATCCAGTGA-3′, 5′-GTCTGGGGAGGGACTTTGTA-3′) were PCR amplified from genomic DNA isolated from cultured human keratinocytes (Gentra Puregene, Qiagen GmbH) with the Primer3-designed oligonucleotides listed in brackets. The VDR DNA-binding domain (DBD, AA 16–125) (Sone et al. 1991) was shuttled into recombinational cloning compatible destination vector pDEST 17 (Life Technologies), transformed into Escherichia coli BL21 (DE3) cells, expressed via 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) induction, and then purified as recombinant hexahistidine-tagged fusion protein by nickel affinity chromatography following common procedures. To investigate binding of VDR-DBD to amplified promoters, 600 ng of respective PCR products were incubated with recombinant DBD in 100 mM Tris buffer in a total reaction volume of 20 μl for 5 min and subsequently separated on a 1% agarose gel in 1× TAE buffer. Samples were visualized for apparent electrophoretic mobility shifts in relation to a previously described osteopontin DR3 repeat (Hijiya et al. 1994) positive control (5′-CGCAGAGCATTTGCATCTAA-3′, 5′-GGTTCTGAATTCCGCTGTGT-3′) plus four analogously treated negative control DNA fragments (designated as C7, D1, A12 and G8) containing no definite VDRE. Band intensities were densitometrically quantified via ImageJ software (http://rsbweb.nih.gov/ij/docs/index.html). Full paper
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