Flag标签系统是用于表达、纯化以及检测融合蛋白的表达系统，广泛应用于Western blotting、免疫细胞组化、免疫共沉淀、流式细胞术、蛋白纯化以及蛋白相互作用、蛋白分离等多个研究领域。Flag标签是一个与重组蛋白相融合的由8个亲水氨基酸（Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys）组成的多肽片断。FLAG标签只有8个氨基酸组成，因此它不会占据其他表位与结合域，避免导致融合蛋白的功能、分泌性以及转运发生改变。由于它的亲水性特点，FLAG标签倾向于定位在融合蛋白表面，因此更易与抗体结合以及被肠激酶酶解。
由于Flag标签可位于蛋白质的C端或N端，这一系统已用于各种细胞类型，包括细菌、酵母和哺乳细胞。由于Flag标签系统的纯化条件是非变性的，因此可以纯化所有有活性的融合蛋白。
有两种商品化的抗体MI和 M2，可识别FLAG标记物。Mi抗体的结合要求表位是氨基末端，因而仅适用于将标记物连接到蛋白的氨基末端。二价阳离子(最好为Ca2+)可增强MI抗 体与FLAG标记物的结合，因此金属螯合剂，如EDTA可引起其相互作用完全或部分破坏，可在很温和的条件下纯化。
M2抗体的存在使该系统显示出更大的应用价值，因为FLAG无论是处于蛋白分子中间
或羧基末端，该抗体可识别相同的FLAG表位。M2对FLAG表位的识别不需要二价阳离子的存在，并且可以通过游离的多肽竞争性地将FLAG标记蛋白从M2分子中温和洗脱下来。
多种FLAG标记蛋白可保留全部的生化活性(如转录因子、生长因子和酶)，这些标记物亦广泛应用于细胞染色、免疫印迹和免疫沉淀试验中。由于已有各种载体和检测试剂出售，包括抗体亲和树脂和直接标记抗体的商品，使其成为人们常选用的系统。
由于对抗-FLAG抗体的特异性进行了更详尽的研究，使得可以采用其他替代序列(Slootstraetal．1997)和更短部分(Knappik和 Pluckthunl994)作为标记物。抗-FLAG抗体产生的本底一般非常低，但M2抗体至少可与一种细胞蛋白发生交叉反应(Schafer和 Braunl995)。
三大内参：beta Actin，肌动蛋白，细胞骨架蛋白。它们在各组织和细胞中的表达相对恒定，在检测蛋白的表达水平变化时常用它来做参照物。beta Actin 作为内参是得到公认的，针对大多数组织和细胞来说的，它广泛分布于细胞质内，表达量非常丰富。但是在一些少量特殊的情况下比如脂肪组织和细胞内，beta Actin 的表达量就很少。GAPDH，甘油醛-3-磷酸脱氢酶，该酶是糖酵解反应中的一个酶，几乎在所有组织细胞中都高水平表达，在同种细胞或组织中的表达量一般是恒定的，且很少受外部诱导物的影响。但比如缺氧和糖尿病等疾病存在下，会增加 GAPDH 在特定细胞和组织中的表达。
作为内参抗体，beta Tubulin 表达通常不会发生改变，因此被广泛用于 Western Blot 内参，也常被用于免疫染色观察细胞的微管结构。
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The Flag tag system is an expression system for expressing, purifying and detecting fusion proteins, which is widely used in Western blotting, immunocytochemistry, immunoprecipitation, flow cytometry, protein purification, protein interaction, protein separation and other research fields. The Flag tag is a
polypeptide fragment composed of eight hydrophilic amino acids (ASP-Tyr-Lys-ASP-ASP-ASP-ASP-Lys) fused with recombinant protein. The FLAG tag consists of only 8 amino acids, so it will not occupy other epitopes and binding domains, which will avoid changing the function, secretion and transport of the fusion protein. Because of its hydrophilicity, the FLAG tag tends to be located on the surface of fusion protein, so it is easier to combine with antibody and be hydrolyzed by enterokinase.
Since the Flag tag can be located at the C-terminal or N-terminal of protein, this system has been used in various cell types, including bacteria, yeast and mammalian cells. Since the purification conditions of the Flag tag system are non-denatured, all active fusion proteins can be purified.
There are two commercial antibodies MI and M2, which can recognize FLAG markers. The binding of Mi antibody requires the epitope to be amino-terminal, so it is only suitable for attaching the marker to the amino-terminal of protein. Bivalent cations (preferably Ca2+) can enhance the binding between MI antibody and FLAG marker, so metal chelating agents, such as EDTA, can cause complete or partial destruction of their interaction, which can be purified under mild conditions.
The existence of M2 antibody makes this system show more application value, because FLAG can recognize the same FLAG epitope whether it is in the middle or carboxyl end of protein molecule. The recognition of FLAG epitope by M2 does not require the existence of divalent cations, and the FLAG marker protein can be competitively eluted from M2 molecule by free polypeptide.
Many FLAG marker proteins can retain all biochemical activities (such as transcription factors, growth factors and enzymes), and these markers are also widely used in cell staining, western blotting and immunoprecipitation tests. Since various carriers and detection reagents have been sold, including antibody affinity resins and commodities directly labeling antibodies, it has become a system that people often choose.
As the specificity of anti-flag antibody has been studied in more detail, other alternative sequences (Slootstra et al. 1997) and shorter parts (Knappik and Pluckthunl994) can be used as markers. The background produced by anti-flag antibody is generally very low, but M2 antibody can cross-react with at least one cellular protein (Schafer and Braunl995).
Three internal references: beta Actin, actin and cytoskeleton protein. Their expression in various tissues and cells is relatively constant, which is often used as a reference when detecting the change of protein expression level. Beta Actin is recognized as an internal reference. For most tissues and cells, it is widely distributed in cytoplasm and has abundant expression. However, in some special cases, such as adipose tissue and cells, the expression of beta Actin is very small. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, is an enzyme in glycolysis reaction, which is expressed at a high level in almost all tissues and cells, and its expression level in the same kind of cells or tissues is generally constant, and is rarely affected by external inducers. However, diseases such as hypoxia and diabetes will increase the expression of GAPDH in specific cells and tissues.
As an internal reference antibody, the expression of beta Tubulin usually does not change, so it is widely used in Western Blot internal reference, and is also often used in immunostaining to observe the microtubule structure of cells.
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