Reagents > IRDye® 700 HIF-1 Consensus Oligonucleotide
5′ — AGC TTG CCC TAC GTG CTG TCT CAG A — 3′
3′ — TCG AAC GGG ATG CAC GAC AGA GTC T — 5′
Underlined nucleotides are the binding site. IRDye 700 oligonucleotides are supplied as 25 µL of 50 nM (or 50 fmol/µL) double-stranded DNA.
HIF-1 is a transcription factor that regulates celluar responses to hypoxia, which have been heavily implicated in cancer biology and various other pathophysiologies. For this reason, it is a common oligonucleotide used in EMSA assays in basic discovery and therapeutics research.
带下划线的核苷酸是结合位点。 IRDye 700 寡核苷酸以 25 µL 的 50 nM（或 50 fmol/µL）双链 DNA 形式提供。
HIF-1 是一种转录因子，可调节细胞对缺氧的反应，这与癌症生物学和各种其他病理生理学密切相关。 出于这个原因，它是基础发现和治疗研究中 EMSA 检测中常用的寡核苷酸。
You should establish conditions of the binding reaction for each protein-DNA pair. For IRDye 700 HIF-1 oligonucleotide, the following binding reaction is a good starting point:
| Reaction | µL |
|---|---|
| 10X Binding Buffer (100 mM Tris, 500 mM KCI, 10 mM DTT; pH 7.5) | 2 |
| Poly (dl•dC) 1 µg/µL in 10 mM Tris, 1 mM EDTA; pH 7.5 | 1 |
| 25 mM DTT/2.5% Tween® 20 | 2 |
| Water | 13 |
| IRDye 700 HIF-1 | 1 |
| COS-7 (CoCl2 TREATED) nuclear extract – (positive control) (5 µg/µL) in Dilution Buffer (20 mM Hepes (pH 7.9), 100 mM KCI, 1 mM MgCl2, 20% glycerol, 0.5 mM PMSF, and 0.5 mM DTT) | 1 |
| Total | 20 |
After the addition of the DNA to the protein-buffer mix, reactions are incubated to allow protein binding to DNA. A typical incubation condition is 20-30 minutes at room temperature.
Since IRDye infrared dyes are somewhat sensitive to light, it is best to keep binding reactions in the dark during incubation periods (e.g., put the tubes into a drawer or simply cover the rack containing tubes with aluminum foil). After the incubation period, 10X Orange Loading Dye (P/N 927-10100) is added to the binding reaction for electrophoresis.
将 DNA 添加到蛋白质缓冲液混合物中后，孵育反应物以使蛋白质与 DNA 结合。 典型的孵育条件是室温下 20-30 分钟。
由于 IRDye 红外染料对光有些敏感，因此最好在孵育期间将结合反应保持在黑暗中（例如，将试管放入抽屉或简单地用铝箔盖住装有试管的架子）。 孵育期后，将 10X Orange Loading Dye (P/N 927-10100) 添加到结合反应中进行电泳。