NHS (N-HydroxySuccinimide) esters and other activated esters (sulfo-NHS, sulfotetrafluorophenyl – STP) are reactive compounds suitable for the modification of amino groups. NHS is most common type of activated esters.

Usual modifications are fluorescent labels, fluorescence quenchers, and other reporter groups. Alkyne and azido group can be attached using activated esters to adapt biomolecules to Click Chemistry.

Since amino groups are nearly always contained in proteins and peptides, modification of these biopolymers is especially common. Other examples are amino-oligonucleotides, amino-modified DNA, and amino-containing sugars.

The reaction of NHS esters with amines is strongly pH-dependent: at low pH, the amino group is protonated, and no modification takes place. At higher-than-optimal pH, hydrolysis of NHS ester is quick, and modification yield diminishes. Optimal pH value for modification is 8.3-8.5.

Water is most common solvent for the labeling. If NHS ester is poorly soluble, it can be added as a solution in DMSO or DMF to a solution of protein in water, adjusted to pH 8.3-8.5. Note that DMF must not contain amines (and thus should have no odor).

We recommend using the following general protocol for the labeling of biomolecules with NHS esters produced by Lumiprobe.

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 is experimental value for mono-labeling, suitable for many common proteins and peptides. However, in some 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 insulin (molar weight 69300 Dalton) with Cy5 NHS ester (molar weight 592 Dalton), and obtain maximum yield of mono-labeled product, one should use 10 × 3 mg × 592 Da / 69300 Da = 0.26 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 scale is low, use minimal volume (10-20 uL). Higher concentrations (1-10 mg of amino-biomolecule per mL of mixture) are optimal.
3. Dissolve NHS ester in 1/10 reaction volume of DMF or DMSO. Amine-free DMF is preferred solvent. After the reaction, NHS ester can be stored in solution for 1-2 months at -20ºC.
4. Dissolve biomolecule in 9/10 reaction volume of buffer with pH 8.3-8.5.0.1 M Sodium bicarbonate solution has appropriate pH. Other alternatives are 0.1 M Tris buffer (although Tris has amino group, it is hindered and does not react with NHS esters), or 0.1 M phosphate buffer. Note pH is the most important thing.When doing large-scale labeling (hundreds of milligrams of NHS ester), note that the mixture tends to acidify with time because of hydrolysis of NHS ester. Monitor pH, or use more concentrated buffer then.
5. Add NHS ester solution to the solution of biomolecule, and vortex well. Keep on ice overnight, or at room temperature during at least 4 hours.

6. Purify the conjugate using appropriate method: gel-filtration for macromolecules is most universal. Precipitation and chromatography is another alternative. Organic impurities (such as N-hydroxysuccinimide, NHS ester, acid produced by hydrolysis) are almost always easily separated. For proteins and nucleic acids, ethanol or acetone precipitation can be used.

NHS（N-羟基琥珀酰亚胺）酯和其它活化酯（磺基-NHS，磺基四氟苯基-STP）是适合于修饰氨基的反应性化合物。 NHS是最常见类型的活化酯。

通常的修饰是荧光标记，荧光淬灭剂和其他报道基团。炔基和叠氮基可以使用活化酯连接以使生物分子适应Click Chemistry。

由于氨基基本上总是包含在蛋白质和肽中，因此这些生物聚合物的修饰是特别常见的。其它实例是氨基寡核苷酸，氨基修饰的DNA和含氨基的糖。

NHS酯与胺的反应是强烈的pH依赖性的：在低pH下，氨基被质子化，并且不发生修饰。在高于最佳pH时，NHS酯的水解快，修饰产率降低。最佳pH值为8.3-8.5。

水是标记的最常见溶剂。如果NHS酯难溶，可将其作为在DMSO或DMF中的溶液加入到蛋白质在水中的溶液中，调节至pH 8.3-8.5。注意DMF不能含有胺（因此应该没有气味）。

我们建议使用以下一般方案用Lumiprobe生产的NHS酯标记生物分子。

计算所需的NHS酯的量： NHS_ester_weight [mg] = 8×amino_compound_weight [mg]×NHS_ester_molar_weight [Da] / amino_compound_molar_weight [Da]。

8是NHS酯的摩尔过量。它是单标记的实验值，适用于许多常见的蛋白质和肽。然而，在一些情况下，需要使用更少或更多的NHS酯。这取决于蛋白质结构，试剂和溶解度。 Lumiprobe产品的摩尔重量可在相应的产品页面找到。

例如，为了用Cy5NHS酯（摩尔质量592道尔顿）标记3mg胰岛素（摩尔重量69300道尔顿），并获得最大产率的单标记产物，应该使用 10×3mg×592Da / 69300Da = 0.26mg 的Cy5染料NHS酯。

确定反应混合物的体积。标记可以从纳摩尔到几十克的任何比例进行。当秤低时，使用最小体积（10-20 uL）。更高的浓度（1-10mg氨基生物分子/ mL混合物）是最佳的。 将NHS酯溶于1/10反应体积的DMF或DMSO中。无胺的DMF是优选的溶剂。反应后，NHS酯可以在-20℃下在溶液中储存1-2个月。 将生物分子溶解在9/10反应体积的pH 8.3-8.5的缓冲液中。 0.1M碳酸氢钠溶液具有适当的pH。其它替代物是0.1M Tris缓冲液（尽管Tris具有氨基，它受阻并且不与NHS酯反应）或0.1M磷酸盐缓冲液。注意pH是最重要的事情。

当进行大规模标记（数百毫克的NHS酯）时，注意到由于NHS酯的水解，混合物倾向于随时间酸化。监测pH值，或使用更浓的缓冲液。

向生物分子溶液中加入NHS酯溶液，充分涡旋。保持在冰上过夜，或在室温下至少4小时。 使用适当的方法纯化结合物：大分子的凝胶过滤是最普遍的。沉淀和色谱是另一种选择。有机杂质（例如N-羟基琥珀酰亚胺，NHS酯，通过水解产生的酸）几乎总是容易分离。对于蛋白质和核酸，可以使用乙醇或丙酮沉淀。