氨基分子的NHS酯標記

氨基分子的NHS酯標記

Lumiprobe Cy熒光染料使用說明書、Cy熒光染料使用量。

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-羥基琥珀酰亞胺）酯和其它活化酯（磺基-NHS，磺基四氟苯基-STP）是適合于修飾氨基的反應性化合物。 NHS是最常見類型的活化酯。

通常的修飾是熒光標記，熒光淬滅劑和其他報道基團。炔基和疊氮基可以使用活化酯連接以使生物分子適應Click Chemistry。

由于氨基基本上總是包含在蛋白質和肽中，因此這些生物聚合物的修飾是特別常見的。其它實例是氨基寡核苷酸，氨基修飾的DNA和含氨基的糖。

NHS酯與胺的反應是強烈的pH依賴性的：在低pH下，氨基被質子化，并且不發(fā)生修飾。在高于佳pH時，NHS酯的水解快，修飾產率降低。佳pH值為8.3-8.5。

水是標記的最常見溶劑。如果NHS酯難溶，可將其作為在DMSO或DMF中的溶液加入到蛋白質在水中的溶液中，調節(jié)至pH 8.3-8.5。注意DMF不能含有胺（因此應該沒有氣味）。

我們建議使用以下一般方案用Lumiprobe生產的NHS酯標記生物分子。

計算所需的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酯的摩爾過量。它是單標記的實驗值，適用于許多常見的蛋白質和肽。然而，在一些情況下，需要使用更少或更多的NHS酯。這取決于蛋白質結構，試劑和溶解度。 Lumiprobe產品的摩爾重量可在相應的產品頁面找到。

例如，為了用Cy5NHS酯（摩爾質量592道爾頓）標記3mg胰島素（摩爾重量69300道爾頓），并獲得最大產率的單標記產物，應該使用
10×3mg×592Da / 69300Da = 0.26mg
的Cy5染料NHS酯。

確定反應混合物的體積。標記可以從納摩爾到幾十克的任何比例進行。當秤低時，使用最小體積（10-20 uL）。更高的濃度（1-10mg氨基生物分子/ mL混合物）是佳的。
將NHS酯溶于1/10反應體積的DMF或DMSO中。無胺的DMF是優(yōu)選的溶劑。反應后，NHS酯可以在-20℃下在溶液中儲存1-2個月。
將生物分子溶解在9/10反應體積的pH 8.3-8.5的緩沖液中。
0.1M碳酸氫鈉溶液具有適當?shù)膒H。其它替代物是0.1M Tris緩沖液（盡管Tris具有氨基，它受阻并且不與NHS酯反應）或0.1M磷酸鹽緩沖液。注意pH是最重要的事情。

當進行大規(guī)模標記（數(shù)百毫克的NHS酯）時，注意到由于NHS酯的水解，混合物傾向于隨時間酸化。監(jiān)測pH值，或使用更濃的緩沖液。

向生物分子溶液中加入NHS酯溶液，充分渦旋。保持在冰上過夜，或在室溫下至少4小時。
使用適當?shù)姆椒兓Y合物：大分子的凝膠過濾是最普遍的。沉淀和色譜是另一種選擇。有機雜質（例如N-羥基琥珀酰亞胺，NHS酯，通過水解產生的酸）幾乎總是容易分離。對于蛋白質和核酸，可以使用乙醇或丙酮沉淀。