Dye maleimides连接带巯基的蛋白或生物分子

 

马来酰***活性基团的荧光染料是目前和带有巯基的生物分子比如说多肽、蛋白或者小分子做标记或者偶联的***常用的***。

 

Maleimides是一个对thiols有高选择性的亲电化合物，在自然界中基本上不存在Maleimides基团，而有丰富的thiols存在，马来酰***和巯基会作为半胱氨酸残基在多肽和蛋白中相遇，虽然天然的DNA中不含有巯基，但是人工合成的寡核苷酸是非常容易制备出来的。

 

Cy3 maleimides , Cy5 maleimides , Cy7 maleimides标记带有SH的多肽、蛋白还有生物小分子的具体实验方法和实验步骤。

 

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 ***(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 (*** 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.

 

相关***信息：

Cyanine3maleimide

 

Appearance:	red powder
Molecular weight:	615.20
Molecular formula:	C36H43ClN4O3
Solubility:	soluble in organic solvents (***, DMSO, ***ane), insoluble in water
Quality control:	NMR 1H (95%) and 13C, TLC, functional testing
Storage conditi***:	Storage: 24 months at -20°C in the dark.

 

Cyanine5maleimide

 

Appearance:	dark blue powder
Molecular weight:	641.24
Molecular formula:	C38H45ClN4O3
Solubility:	soluble in organic solvents (***, DMSO, ***ane), insoluble in water
Quality control:	NMR 1H (95%) and 13C, TLC, functional testing
Storage conditi***:	Storage: 24 months at -20°C in the dark

 

Cyanine7maleimide

 

Appearance:	green powder
Molecular weight:	707.34
Molecular formula:	C43H51ClN4O3
Solubility:	soluble in organic solvents (DMSO, ***, ***ane), low solubility in water
Quality control:	NMR 1H (95%) and 13C, TLC, functional testing
Storage conditi***:	Storage: 24 months at -20°C in the dark.