一种或化羧基染料CY3/CY5.5/CY7.5 NHS ester标记氨基分子方法

西安瑞禧生物科技有限公司提供一种带NHS活化羧基的荧光CY系列染料用于标记氨基的方法说明

瑞禧生物科技可以提供的活化羧基系列荧光染料包括：

Cyanine3 NHS ester

Cyanine5 NHS ester

Cyanine5.5 NHS ester

Cyanine7 NHS ester

Cyanine7.5 NHS ester

Cy3 NHS ester

Cy5 NHS ester

Cy5.5 NHS ester

Cy7 NHS ester

Cy7.5 NHS ester

标记方法说明如下：

NHS (N-HydroxySuccinimide) esters and otheractivated esters (sulfo-NHS, sulfotetrafluorophenyl-STP) are reactive compoundssuitable for the modification of amino groups. NHS is most common type ofactivated esters.

Usual modifications are fluorescent labels,fluorescence quenchers, and other reporter groups.

Alkyne and azido group can be attachedusing activated esters to adapt biomolecules to Click

Chemistry.

Since amino groups are nearly alwayscontained in proteins and peptides, modification of these

biopolymers is especially common. Otherexamples are amino-oligonucleotides, amino-modified DNA,and amino-containingsugars.

The reaction of NHS esters with amines isstrongly pH-dependent: at low pH, the amino group is

protonated, and no modification takesplace. At higher-than-optimal pH, hydrolysis of NHS ester isquick, andmodification yield diminishes. Optimal pH value for modification is 8.3-8.5.

Water is most common solvent for thelabeling. If NHS ester is poorly soluble, it can be added as a

solution in DMSO or DMF to a solution ofprotein in water, adjusted to pH 8.3-8.5. Note that DMFmust not contain amines(and thus should have no odor).

We recommend using the following generalprotocol for the labeling of biomolecules with NHS esters produced by Ruixibio.

1. Calculate required amount of NHS ester:

NHS_ester_weight [mg] = 8 ×amino_compound_weight [mg] × NHS_ester_molar_weight [Da] /

amino_compound_molar_weight [Da].

8 is molar excess of NHS ester. It isexperimental value for mono-labeling, suitable for many

common proteins and peptides. However, insome cases using less or more NHS ester is required.

It depends on protein structure, reagent,and solubility. Molar weight of Lumiprobe products can

be found on corresponding product pages.

For example, to label 3 mg of BSA (molarweight 69300 Dalton) with Cy5 NHS ester (molar weight 616 Dalton), andobtainmaximum yield of mono-labeled product, one should use 8 × 3 mg × 616 Da / 69300Da = 0.21 mg of Cy5 dye NHS ester.

2. Determine volume of reaction mixture.The labeling can be performed on any scale from

nanomols to dozens of grams. When the scaleis low, use minimal volume (10-20 uL). Higher

concentrations (1-10 mg of amino-biomoleculeper mL of mixture) are optimal.

3. Dissolve NHS ester in 1/10 reactionvolume of DMF or DMSO. Amine-free DMF is preferred

solvent. After the reaction, NHS ester canbe stored in solution for 1-2 months at -20ºC.

4. Dissolve biomolecule in 9/10 reactionvolume of buffer with pH 8.3-8.5.

0.1 M Sodium bicarbonate solution hasappropriate pH. Another alternative is 0.1 M phosphate

buffer. Note pH is the most importantthing. Avoid using buffers containing amines (Tris can

sometimes be used but not recommended).

When doing large-scale labeling (hundredsof milligrams of NHS ester), note that the mixture

tends to acidify with time because ofhydrolysis of NHS ester. Monitor pH, or use more

concentrated buffer then.

5. Add NHS ester solution to the solutionof biomolecule, and vortex well. Keep on ice overnight,

or at room temperature during at least 4hours.

6. Purify the conjugate using appropriatemethod: gel-filtration for macromolecules is most

universal. Precipitation and chromatographyis another alternative. Organic impurities (such as Nhydroxysuccinimide,NHSester, acid produced by hydrolysis) are almost always easily separated.Forproteins and nucleic acids, ethanol or acetone precipitation can be used.