荧光标记肽-荧光标记法的原理

全国荧光标记肽-北京荧光标记法的原理-上海荧光标记技术-天津荧光蛋白标记技术

荧光标记所依赖的化合物称为荧光物质。荧光物质是指具有共轭双键体系化学结构的化合物，受到紫外光或蓝紫光照射时，可激发成为激发态，当从激发态恢复基态时，发出荧光。荧光标记技术指利用荧光物质共价结合或物理吸附在所要研究分子的某个基团上，利用它的荧光特性来提供被研究对象的信息。荧光标记的无放射物污染，操作简便等优点，使得荧光标记物在许多研究领域的应用日趋广泛。

荧光标记物质在蛋白的功能研究、药物筛选等领域也有着广泛的应用。人们利用利用荧光标记的多肽来检测目标蛋白的活性，并将其发展的高通量活性筛选方法应用于疾病ZL靶点蛋白的药物筛选和药物开发（例如，各种激酶、磷酸酶、肽酶等）。因此，多肽的荧光修饰，同样是多肽合成领域的重要内容。

下面是一些常用的多肽修饰荧光物质：

下面是一些荧光物质的激发光波长和发射光波长

1.FITC修饰

异硫氰酸荧光素（FITC）具有比较高的活性，通常来说，在固相合成过程中引入该种荧光基团相对于其他荧光素要更容易，并且反应过程中不需要加入活化试剂。

我们公司合成的FITC修饰的多肽通常主要有两种形式：

（1）在整条肽链末端接入FITC，并且在FITC之前接入一分子的Acp（6-氨基己酸），也称烷基间隔器。反应中FITC与肽链上裸露的-NH2反应，Acp的接入提供了六个碳的直链空间，大大降低了反应的空间位阻，提高了反应效率，降低了反应难度。其次，FITC还与多肽结构中的-SH，侧链-NH2反应，Acp的加入也降低了这种副反应发生的可能。此外，多肽在酸性环境条件下切割时，在N端接入FITC的多肽需要经历环化作用来形成荧光素，这种过程通常都会伴随Z后一个氨基酸的切除，而烷基间隔器Acp的接入就避免了这一情况的发生。

（2）在整条肽中的某个Lys侧链接入FITC，Lys侧链为末端为-NH2的四碳直链烷基，直接起到了降低空间位阻的作用。这种修饰方式能够灵活的在整条肽中任何位置进行FITC修饰，而不仅仅局限于末端。

我们所采用的FITC修饰多肽的两种形式，都具有操作简便，成功率高，容易分离纯化等优点。

2.AMC修饰

7-氨基-4-甲基香豆素（AMC）是一种应用广泛的荧光标记试剂，例如，酶的痕量测定，酶的鉴定，激光染料的制备等多种用途，此外，C端用香豆素修饰的泛素分子也是研究蛋白质泛素化过程的重要探针。

与其他荧光染料不同的是，AMC修饰多肽分子是从C端进行：（1）AMC与肽链C端**个氨基酸反应;（2）固相合成整条肽链（从第二个氨基酸开始），并且保留整条肽链的侧链保护基和Z后一个氨基保护基；（3）液相缩合AA-AMC与全保护的肽链；（4）切除保护基，完成肽链的修饰。

国肽生物提供5(6)-FAM，FITC，CY5，RhodamineB，PNA，EDNAS/dabcyl，Biotin等各种修饰的高质量多肽。国肽生物具有成熟的荧光标记多肽技术，优良的纯化生产工艺，定制荧光修饰的多肽，国肽生物是值得信任的品牌。

成功案例：

序列Cy5-betaA-YNDEDPEKEKRIKELELLLMSTENELKGQQAL，CY5进行修饰。

HPLC分析：

MS分析：

合肥国肽生物官网：http://www.bankpeptide.com

欢迎咨询服务热线：17718122684；17718122172；17730030476；17718122397

翻译：

translation:

The compound on which the fluorescent label is dependent is called a fluorescent substance. A fluorescent substance refers to a compound having a chemical structure of a conjugated double bond system, and when excited by ultraviolet light or blue-violet light, it can be excited to become an excited state, and when it is restored from the excited state, it emits fluorescence. Fluorescent labe领 technology refers to the use of a fluorescent substance to covalently bind or physically adsorb on a certain group of a molecule to be studied, and to use its fluorescent properties to provide information on the object to be studied. Fluorescently labeled non-radioactive contamination, easy to operate, etc., make fluorescent labels widely used in many research fields.

Fluorescent labe领 substances are also widely used in the fields of functional research and drug screening of proteins. The use of fluorescently labeled polypeptides to detect the activity of a target protein and its development of high-throughput activity screening methods for drug screening and drug development of disease-targeted proteins (eg, various kinases, phosphatases, peptidases) Wait). Therefore, the fluorescent modification of the polypeptide is also an important content in the field of polypeptide synthesis.

Here are some commonly used peptide-modified fluorescent substances:

The following are the excitation light wavelengths and emission wavelengths of some fluorescent substances.

1.FITC modification

Fluorescein isothiocyanate (FITC) has a relatively high activity. Generally, it is easier to introduce such a fluorophore in the solid phase synthesis process than other fluorescein, and it is not necessary to add an activating reagent during the reaction.

The FITC-modified peptides synthesized by our company usually have two main forms:

(1) APTC is inserted at the end of the entire peptide chain, and a molecule of Acp (6-aminocaproic acid), also referred to as an alkyl spacer, is inserted before FITC. In the reaction, FITC reacts with the exposed -NH2 on the peptide chain. The access of Acp provides a linear space of six carbons, which greatly reduces the steric hindrance of the reaction, improves the reaction efficiency, and reduces the reaction difficulty. Secondly, FITC also reacts with -SH and side chain -NH2 in the structure of the polypeptide. The addition of Acp also reduces the possibility of this side reaction. In addition, when the polypeptide is cleaved under acidic environmental conditions, the polypeptide that is inserted into the FITC at the N-terminus needs to undergo cyclization to form fluorescein. This process is usually accompanied by the excision of the last amino acid, and the access of the alkyl spacer Acp. This avoids this happening.

(2) A single Lys side of the entire peptide is linked into FITC, and the Lys side chain is a four-carbon linear alkyl group having a terminal -NH2, which directly acts to reduce steric hindrance. This modification allows for flexible FITC modification anywhere in the entire peptide, not just the ends.

The two forms of FITC modified polypeptides we use have the advantages of simple operation, high success rate, easy separation and purification.

2. AMC modification

7-Amino-4-methylcoumarin (AMC) is a widely used fluorescent labe领 reagent, for example, trace determination of enzymes, identification of enzymes, preparation of laser dyes, etc., in addition, C-end The coumarin-modified ubiquitin molecule is also an important probe for studying the process of protein ubiquitination.

Unlike other fluorescent dyes, AMC-modified polypeptide molecules are carried out from the C-terminus: (1) AMC reacts with the first amino acid of the C-terminus of the peptide chain; (2) Solid phase synthesizes the entire peptide chain (starting with the second amino acid) And retaining the side chain protecting group and the last amino protecting group of the entire peptide chain; (3) liquid phase condensing AA-AMC with a fully protected peptide chain; (4) excising the protecting group to complete modification of the peptide chain.

The peptidomimetic provides various modified high quality polypeptides such as 5(6)-FAM, FITC, CY5, Rhodamine B, PNA, EDNAS/dabcyl, Biotin. National peptide organisms have mature fluorescent-labeled peptide technology, excellent purification production process, custom fluorescent modified peptides, and national peptide organisms are trustworthy brands.

success case:

The sequence Cy5-betaA-YNDEDPEKEKRIKELELLLMSTENELKGQQAL, CY5 was modified.

HPLC analysis:

MS analysis:

Bankpeptide biological technology co.,LTD: http://www.bankpeptide.com

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