Dye maleimides連接帶巰基的蛋白或生物分子
馬來酰亞胺活性基團(tuán)的熒光染料是目前和帶有巰基的生物分子比如說多肽、蛋白或者小分子做標(biāo)記或者偶聯(lián)的常用的試劑。
Maleimides是一個(gè)對(duì)thiols有高選擇性的親電化合物，在自然界中基本上不存在Maleimides基團(tuán)，而有豐富的thiols存在，馬來酰亞胺和巰基會(huì)作為半胱氨酸殘基在多肽和蛋白中相遇，雖然天然的DNA中不含有巰基，但是人工合成的寡核苷酸是非常容易制備出來的。
Cy3 maleimides , Cy5 maleimides , Cy7 maleimides標(biāo)記帶有SH的多肽、蛋白還有生物小分子的具體實(shí)驗(yàn)方法和實(shí)驗(yàn)步驟。
1. Dissolve the protein or other moleculecontaining thiol to be labeled in degassed buffer (PBS, Tris, HEPES are good,although others buffers containing no thiols can be used) at pH 7-7.5 inplastic vial. Buffer can be degassed by applying vacuum on it for severalminutes, or by bubbling through inert gas (nitrogen, argon, or helium). Forproteins, good concentration is between 1-10 mg/mL.
2. Add an excess of TCEP(tris-carboxyethylphosphine) reagent to reduce disulfide bonds, flush withinert gas, and close. 100x molar excess of TCEP is fine. Keep the mixture for20 minutes at room temperature.
3. Dissolve maleimide in DMSO or fresh DMF(1-10 mg in 100 uL).
4. Add dye solution to thiol solution (20xfold excess of dye), flush vial with inert gas, and close tightly. 5. Mixthoroughly, and keep overnight at room temperature, or 4 Celsius. 6. Purify bygel filteration, HPLC, FPLC, or electrophoresis
For maleimides with poor aqueoussolubility, like most dye maleimides, we recommend use of cosolvent (DMF orDMSO). Maleimides with good aqueous solubility (like sulfo-Cy maleimides) canbe dissolved in water. If precipitation occurs, increase content of organicco-solvent in the mixture to achieve better labeling.
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相關(guān)試劑信息：
Cyanine3 maleimide
Appearance: | red powder |
Molecular weight: | 615.20 |
Molecular formula: | C36H43ClN4O3 |
Solubility: | soluble in organic solvents (DMF, DMSO, dichloromethane), insoluble in water |
Quality control: | NMR 1H (95%) and 13C, TLC, functional testing |
Storage conditions: | Storage: 24 months at -20°C in the dark. |
Cyanine5 maleimide
Appearance: | dark blue powder |
Molecular weight: | 641.24 |
Molecular formula: | C38H45ClN4O3 |
Solubility: | soluble in organic solvents (DMF, DMSO, dichloromethane), insoluble in water |
Quality control: | NMR 1H (95%) and 13C, TLC, functional testing |
Storage conditions: | Storage: 24 months at -20°C in the dark |
Cyanine7 maleimide
Appearance: | green powder |
Molecular weight: | 707.34 |
Molecular formula: | C43H51ClN4O3 |
Solubility: | soluble in organic solvents (DMSO, DMF, dichloromethane), low solubility in water |
Quality control: | NMR 1H (95%) and 13C, TLC, functional testing |
Storage conditions: | Storage: 24 months at -20°C in the dark. |
