此页面上的某些内容不提供英文版本，默认显示为当前可用的语言。

NHS Ester Labeling of Biomolecules Containing Primary Amino Groups

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

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

Especially common are modifications of proteins and peptides since they nearly always contain amino groups. Other examples are modifications of amino-oligonucleotides, amino-modified DNA, and amino-containing sugars.

The reaction of NHS esters with amino groups is strongly pH-dependent: at low pH, the amino group is protonated, and the modification does not occur. At higher-than-optimal pH, hydrolysis of NHS ester is quick, and the yield of modified molecule decreases. The optimal pH value for modification is 8.3-8.5.

Water is the most common solvent used to dissolve NHS esters for biomolecule labeling. If NHS ester is poorly soluble in aqueous solutions, it can be at first dissolved in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and then added to a solution of protein in a buffer with pH 8.3-8.5. Note that DMF can degrade into dimethylamine, which has a fishy smell and is able to react with activated esters. Therefore, to label biomolecules, it is important to use high-quality DMF that contains no dimethylamine and has no fishy odor.

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

Calculate the required amount of NHS ester using the formula below or our protein labeling calculator:
NHS_ester_weight [mg] = 8 × amino_compound_weight [mg] × NHS_ester_molar_weight [Da] / amino_compound_molar_weight [Da].
8 is a molar excess of NHS ester. It is an empirical value for mono-labeling, suitable for many common proteins and peptides. However, in some cases using less or more NHS ester can increase the yield of the modified biomolecule. It depends on protein structure, reagent used for labeling, and its solubility. The molar weight of Lumiprobe products can be found on the corresponding product pages.
For example, to label 3 mg of BSA (molar weight 66500 Dalton) with Cy5 NHS ester (molar weight 616 Dalton), and obtain maximum yield of mono-labeled product, one should use 8 × 3 mg × 616 Da / 66500 Da = 0.22 mg of Cy5 dye NHS ester.
Calculate the volume of the reaction mixture. The optimal biomolecule concentration in labeling reaction is 1-10 mg per mL. The labeling can be performed on any scale, from nanomols to dozens of grams. If you label a low amount of biomolecule, keep the volume of the reaction mixture at a minimum (10-20 uL).
Dissolve NHS ester in 1/10 reaction volume of water, DMF, or DMSO. Dimethylamine-free DMF or water are preferred solvents. NHS ester dissolved in DMF can be stored in solution for 1-2 months at −20ºC. The aqueous solution of NHS ester should be used immediately after preparation.
Dissolve biomolecule in 9/10 reaction volume of buffer with pH 8.3-8.5.
0.1 M Sodium bicarbonate solution has appropriate pH. Another alternative is 0.1 M phosphate buffer. Note that pH is the most important factor for successfully labeling biomolecule with NHS ester. Avoid buffers containing amines (Tris-based buffers can sometimes be used but are not recommended. Tris contains amino group, but its affinity to activated esters is low).
When doing large-scale labeling (using hundreds of milligrams of NHS ester), note that the mixture tends to acidify with time because of the hydrolysis of NHS ester. Monitor pH, or use a more concentrated buffer then.
Add NHS ester solution to the solution of biomolecule, and vortex well. Keep on ice overnight, or at room temperature from 1 to 4 hours.
Purify the conjugate using the appropriate method: gel-filtration for macromolecules is the most common. Chromatography is another alternative. For proteins and nucleic acids, ethanol or acetone precipitation can be used. Organic impurities (such as N-hydroxysuccinimide, NHS ester, and acid produced by hydrolysis) are almost always easily separated by these methods.

相关产品

3-马来酰亚胺丙酸NHS-酯 (3-Maleimidopropionic acid NHS-ester)

具有胺反应性琥珀酰亚胺酯和巯基反应性的马来酰亚胺基团的双功能交联剂。

显示价格

目录号	数量	价格	提前通知时间	购买此产品
3419-100mg	100 mg	–	现货
3419-500mg	500 mg	$110	现货

样品请求

Found better price? Let us know and we will propose the way forward!

AF 488 TFP 酯 (AF 488 TFP ester)

绿色荧光染料 AF 488 的四氟苯酯，用于标记蛋白质、多肽和修饰寡核苷酸中的氨基。

显示价格

目录号	数量	价格	提前通知时间
118T0	1 mg	$165	21 天
218T0	5 mg	$510	现货
418T0	25 mg	$1650	现货

目录号	数量	价格	提前通知时间
1C920	1 mg	$110	现货
2C920	5 mg	$640	现货
4C920	25 mg	$1640	现货
5C920	50 mg	$2090	现货
6C920	100 mg	$2840	现货

目录号	数量	价格	提前通知时间
14420	1 mg	$125	现货
24420	5 mg	$260	现货
44420	25 mg	$510	现货
54420	50 mg	$895	现货
64420	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
34720	10 mg	$80	现货
44720	25 mg	$110	现货
54720	50 mg	$135	现货
64720	100 mg	$210 $178	现货
94720	1 g	$1000 $970	现货
K4720	5 g	$3570 $2990	现货

目录号	数量	价格	提前通知时间
2584-5mg	5 mg	–	现货
2584-10mg	10 mg	$90	现货
2584-25mg	25 mg	$180	现货
2584-100mg	100 mg	$450	现货
2584-500mg	500 mg	$1700	5 天
2584-1g	1 g	$3000	现货
2584-5g	5 g	请查询	现货

目录号	数量	价格	提前通知时间
4245-100mg	100 mg	$125	现货
4245-500mg	500 mg	$525	现货
4245-1g	1 g	$990	现货

目录号	数量	价格	提前通知时间
2782-100mg	100 mg	$110	现货
2782-500mg	500 mg	$485	现货
2782-1g	1 g	$895	现货

目录号	数量	价格	提前通知时间
22D20	5 mg	–	现货
42D20	25 mg	$315	现货
52D20	50 mg	$580	现货
62D20	100 mg	$885	现货

目录号	数量	价格	提前通知时间
18120	1 mg	$110	现货
28120	5 mg	$210	现货
48120	25 mg	$350	现货
58120	50 mg	$595	现货
68120	100 mg	$790	5 天

目录号	数量	价格	提前通知时间
3056-10mg	10 mg	$145	现货
3056-25mg	25 mg	$225	现货
3056-100mg	100 mg	$550	现货

目录号	数量	价格	提前通知时间
1V220	1 mg	$125	现货
2V220	5 mg	$325	现货
4V220	25 mg	$510	现货
5V220	50 mg	$895	现货
6V220	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
53720	50 mg	$110	现货
63720	100 mg	$210	现货
73720	1 g	$950	现货
83720	5 g	请查询	现货

目录号	数量	价格	提前通知时间
41720	25 mg	$95	现货
51720	50 mg	$180	现货
61720	100 mg	$290	现货
91720	1 g	请查询	现货

目录号	数量	价格	提前通知时间
30720	10 mg	$110	现货
40720	25 mg	$210	现货
50720	50 mg	$410	现货
60720	100 mg	$595	10 天

目录号	数量	价格	提前通知时间
13320	1 mg	$91	现货
23320	5 mg	$119	现货
43320	25 mg	$345	现货
53320	50 mg	$557	现货
63320	100 mg	$890	现货

搜索产品

NHS Ester Labeling of Biomolecules Containing Primary Amino Groups

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

相关产品

搜索产品

免费

无手续费！

帮助中心

更多信息

分析证书

Lumiprobe Corporation (美洲)