Cy7 maleimide染料標(biāo)記巰基化生物分子實(shí)驗(yàn)方案

Cy7馬來(lái)酰亞胺是一種選擇性和的低水溶性熒光染料。該標(biāo)記試劑可用于將Cy7熒光團(tuán)附著于含有半胱氨酸殘基的蛋白質(zhì)和肽，以及其它硫醇分子（如含巰基的寡核苷酸）。在標(biāo)記反應(yīng)之前，胱氨酸應(yīng)該用TCEP（三羧乙基膦）還原。對(duì)于生物分子標(biāo)記，標(biāo)記試劑具有低的水溶解度，使用有機(jī)共溶劑來(lái)溶解該分子是反應(yīng)所必需的。先，Cyanine染料應(yīng)溶解在有機(jī)溶劑中，然后在適當(dāng)?shù)乃跃彌_液中添加到生物分子溶液中。

生物數(shù)據(jù)

在10μg ML-1濃度下，用Cy3馬來(lái)酰亞胺標(biāo)記MSNs，命名為MSN-CY3S。MSN-CY3S與MEF MEGFP在培養(yǎng)基中的玻璃蓋玻片上孵育2小時(shí)，以進(jìn)一步觀察內(nèi)的MSN位置，我們使用分辨率顯微鏡（3D SIM）。視覺(jué)顯微鏡圖像結(jié)果證實(shí)MSNs被MEF MEGFP內(nèi)化。

用Cy3/ Cy5/ Cy7 maleimide 染料標(biāo)記蛋白質(zhì)、肽和其他巰基化生物分子

Protocol: Maleimide labeling of proteins and other thiolated biomolecules

Reaction of thiols with maleimides is a process which is widely used for bioconjugation and labeling of biomolecules including proteins and peptides. It proceeds according to the following scheme:

Thiol maleimide reaction scheme

Maleimides are electrophilic compounds which show high selectivity towards thiols. While maleimides hardly ever occur in nature, thiols are very abundant. They are encountered in proteins and peptides as cysteine residues. Although natural DNA does not contain thiols, synthetic oligonucleotides with thiol groups can be easily prepared.

Thiols are prone to oxidative dimerization with the formation of disulfide bonds. Cysteine residues thus form disulfide bridges, which stabilize protein tertiary structures. Disulfides do not react with maleimides. Therefore, it is necessary to reduce disulfides prior to the conjugation, and to exclude oxygen from the reaction.

Conjugation protocol depends on the solubility of the starting components. For compounds with low aqueous solubility, like most fluorescent dye maleimides, use of organic co-solvent, such as DMSO or DMF, is essential.

We recommend using the following general protocol for the labeling of proteins, peptides, and other thiolated biomolecules with APExBIO dye maleimides.

1. Dissolve the protein or other molecule containing 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 in plastic vial. Buffer can be degassed by applying vacuum on it for several minutes, or by bubbling through inert gas (nitrogen, argon, or helium). For proteins, good concentration is between 1-10 mg/mL.

2.  2. Add an excess of TCEP (tris-carboxyethylphosphine) reagent to reduce disulfide bonds, flush with inert gas, and close. 100x molar excess of TCEP is fine. Keep the mixture for 20 minutes at room temperature.

3. 3. Dissolve maleimide in DMSO or fresh DMF (1-10 mg in 100 uL).

4.  4. Add dye solution to thiol solution (20x fold excess of dye), flush vial with inert gas, and close tightly.

5.  5. Mix thoroughly, and keep overnight at room temperature, or 4 Celsius. 6. Purify by gel filteration, HPLC, FPLC, or electrophoresis.

For maleimides with poor aqueous solubility, like most dye maleimides, we recommend use of co-solvent (DMF or DMSO). Maleimides with good aqueous solubility (like sulfo-Cy maleimides) can be dissolved in water. If precipitation occurs, increase content of organic co-solvent in the mixture to achieve better labeling. Dialysis is recommended as a means of purification only for maleimides with good aqueous solubility.

用Cy3/ Cy5/ Cy7 maleimide 染料標(biāo)記蛋白質(zhì)、肽和其他巰基化生物分子

我們建議以下方案將Cy3/Cy5/Cy7maleimide 馬來(lái)酰亞胺與蛋白質(zhì)，肽，和其他巰基化生物分子。

具體反應(yīng)步驟:

1.將含有巰基的蛋白質(zhì)或其它分子溶解在脫氣緩沖液中（PBS、TIS、HEPES）是好的，但在塑料瓶中的pH值為7.7.5時(shí)，可以使用其它不含硫醇的緩沖液。緩沖器可以通過(guò)在其上施加真空幾分鐘，或者通過(guò)惰性氣體（氮?dú)?、氬氣或氦氣）鼓泡?lái)脫氣。對(duì)于蛋白質(zhì)，的濃度在1-10mg/mL之間。

2.加入過(guò)量的TCEP（TIS羧乙基膦）試劑，以減少二硫鍵，用惰性氣體沖洗，并關(guān)閉。100X摩爾過(guò)量的TCEP是的。室溫下混合20分鐘。

3.將馬來(lái)酰亞胺溶解在DMSO或鮮DMF中（1-10毫克在100μL）。

4.將染料溶液加入硫醇溶液（染料20X倍過(guò)量），用惰性氣體沖洗小瓶，并緊密地封閉。

5.*混合，在室溫或4攝氏度下過(guò)夜。

6.通過(guò)凝膠過(guò)濾、HPLC、FPLC或電泳純化。

對(duì)于水溶性差的馬來(lái)酰亞胺，與大多數(shù)染料馬來(lái)酰亞胺一樣，我們使用有機(jī)溶劑（DMF或DMSO）。具有水溶性的馬來(lái)酰亞胺（如磺?；R來(lái)酰亞胺Sulfo-Cyanine5maleimide）可以溶解在水中。如果發(fā)生沉淀，增加有機(jī)共溶劑在混合物中的含量，以實(shí)現(xiàn)好的標(biāo)記。透析是一種對(duì)具有水溶性的馬來(lái)酰亞胺進(jìn)行純化的方法。

Cy3馬來(lái)酰亞胺Cyanine3 maleimide, 10mg

Cy3馬來(lái)酰亞胺Cyanine3 maleimide, 25 mg

Cy3馬來(lái)酰亞胺Cyanine3 maleimide, 50 mg

Cy3馬來(lái)酰亞胺Cyanine3 maleimide, 100 mg

Cy3.5馬來(lái)酰亞胺Cyanine3.5 maleimide, 10mg

Cy3.5馬來(lái)酰亞胺Cyanine3.5 maleimide, 25 mg

Cy3.5馬來(lái)酰亞胺Cyanine3.5 maleimide, 50 mg

Cy3.5馬來(lái)酰亞胺Cyanine3.5 maleimide, 100 mg

Cy5馬來(lái)酰亞胺Cyanine5 maleimide, 10mg

Cy5馬來(lái)酰亞胺Cyanine5 maleimide, 25 mg

Cy5馬來(lái)酰亞胺Cyanine5 maleimide, 50 mg

Cy5馬來(lái)酰亞胺Cyanine5 maleimide, 100 mg

Cy5.5馬來(lái)酰亞胺Cyanine5.5 maleimide, 10 mg

Cy5.5馬來(lái)酰亞胺Cyanine5.5 maleimide, 25 mg

Cy5.5馬來(lái)酰亞胺Cyanine5.5 maleimide, 50 mg

Cy5.5馬來(lái)酰亞胺Cyanine5.5 maleimide, 100 mg

Cy7馬來(lái)酰亞胺Cyanine7 maleimide, 10mg

Cy7馬來(lái)酰亞胺Cyanine7 maleimide, 25 mg

Cy7馬來(lái)酰亞胺Cyanine7 maleimide, 50 mg

Cy7馬來(lái)酰亞胺Cyanine7 maleimide, 100 mg

Cy7.5馬來(lái)酰亞胺Cyanine7.5 maleimide, 10 mg

Cy7.5馬來(lái)酰亞胺Cyanine7.5 maleimide, 25 mg

Cy7.5馬來(lái)酰亞胺Cyanine7.5 maleimide, 50 mg

Cy7.5馬來(lái)酰亞胺Cyanine7.5 maleimide, 100 mg

磺酸基-Cy3 馬來(lái)酰亞胺Sulfo-Cyanine3 maleimide, 10 mg

磺酸基-Cy3 馬來(lái)酰亞胺Sulfo-Cyanine3 maleimide, 25 mg

磺酸基-Cy3 馬來(lái)酰亞胺Sulfo-Cyanine3 maleimide, 50 mg

磺酸基-Cy3.5 馬來(lái)酰亞胺Sulfo-Cyanine3 maleimide, 10 mg

磺酸基-Cy3.5 馬來(lái)酰亞胺Sulfo-Cyanine3 maleimide, 25 mg

磺酸基-Cy3.5 馬來(lái)酰亞胺Sulfo-Cyanine3 maleimide, 50 mg

磺酸基-Cy3.5 馬來(lái)酰亞胺Sulfo-Cyanine3 maleimide, 100 mg

磺酸基-Cy5 馬來(lái)酰亞胺Sulfo-Cyanine5 maleimide, 10 mg

磺酸基-Cy5 馬來(lái)酰亞胺Sulfo-Cyanine5 maleimide, 25 mg

磺酸基-Cy5 馬來(lái)酰亞胺Sulfo-Cyanine5 maleimide, 50 mg

磺酸基-Cy5 馬來(lái)酰亞胺Sulfo-Cyanine5 maleimide, 100 mg

磺酸基-Cy5.5 馬來(lái)酰亞胺Sulfo-Cyanine5.5 maleimide, 10 mg

磺酸基-Cy5.5 馬來(lái)酰亞胺Sulfo-Cyanine5.5 maleimide, 25 mg

磺酸基-Cy5.5 馬來(lái)酰亞胺Sulfo-Cyanine5.5 maleimide, 50 mg

磺酸基-Cy5.5 馬來(lái)酰亞胺Sulfo-Cyanine5.5 maleimide, 100 mg

磺酸基-Cy7 馬來(lái)酰亞胺Sulfo-Cyanine7 maleimide, 10 mg

磺酸基-Cy7 馬來(lái)酰亞胺Sulfo-Cyanine7 maleimide, 25 mg

磺酸基-Cy7 馬來(lái)酰亞胺Sulfo-Cyanine7 maleimide, 50 mg

磺酸基-Cy7 馬來(lái)酰亞胺Sulfo-Cyanine7 maleimide, 100 mg

磺酸基-Cy7.5 馬來(lái)酰亞胺Sulfo-Cyanine7.5 maleimide, 10 mg

磺酸基-Cy7.5 馬來(lái)酰亞胺Sulfo-Cyanine7.5 maleimide, 25 mg

磺酸基-Cy7.5 馬來(lái)酰亞胺Sulfo-Cyanine7.5 maleimide, 50 mg

磺酸基-Cy7.5 馬來(lái)酰亞胺Sulfo-Cyanine7.5maleimide, 100 mg