Reagents > IRDye® 700 CREB Consensus Oligonucleotide,IRDye® 700 CREB 共有寡核苷酸
5′ — AGA GAT TGC CTG ACG TCA GAG AGC TAG — 3′
3′ — TCT CTA ACG GAC TGC AGT CTC TCG ATC — 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.
CREB proteins are cellular transcription factors activated by phosphorylation of various kinases that bind to certain DNA sequences. For this reason, it is a common oligonucleotide used in EMSA to assess protein binding.
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。
CREB 蛋白是由与某些 DNA 序列结合的各种激酶的磷酸化激活的细胞转录因子。 出于这个原因，它是 EMSA 中用于评估蛋白质结合的常见寡核苷酸。
You should establish conditions of the binding reaction for each protein-DNA pair. For IRDye 700 CREB 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 |
| 1% NP-40 | 1 |
| Water | 12 |
| IRDye 700 CREB | 1 |
| PC12 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。