FLAG tag Peptide is an 8-peptide (Asp-Tyr-lys-Asp-Asp-Asp-ASP-ASP-Lys) containing an intestinal kinase restriction site. FLAG Tag peptide fusion system is used in immunoaffinity chromatography for protein identification and purification. FLAG tag Peptide label peptides allow elution under non-denatured conditions[1,6]. The FLAG tag Peptide can be fused to either the N- or C-terminus of a given fusion protein.
The FLAG tag Peptide marker peptide fusion system comprises a unique and widely useful technique for protein identification and purification. When covalently attached to a solid support, the anti-Flag M1 antibody can be used for the rapid purification of FLAG tag Peptide fusion proteins in a mild, calcium-dependent affinity chromatography procedure. FLAG tag Peptide fusion proteins are typically purified to homogeneity in a single step, starting from a crude cell homogenate or supernatant, without ever exposing the protein to conditions other than physiological saline at pH 7.2 (with calcium or EDTA). The entire purification process can be carried out within several hours[2-3].
The N-terminal FLAG tag Peptide fusion protein can be purified by agarose affinity gel[5]. A short-cut protein purification procedure, where mechanical cell disintegration and affinity purification by immobilization of the anti-Flag M1 antibody on magnetic glass beads are combined. The modified glass beads were added to yeast cells and mixed on a laboratory vortex mixer. The magnetic glass beads capture the target protein, while the yeast cells are disrupted. By making contact with a stationary magnet, the magnetic glass beads can very efficiently be separated from yeast cell debris. Chelating agents, such as EDTA, were capable of eluting the FLAG tag Peptide fusion proteins from the magnetic glass beads[4].
References:
[1]. Einhauer A, Jungbauer A. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods. 2001 Oct 30;49(1-3):455-65. doi: 10.1016/s0165-022x(01)00213-5. PMID: 11694294.
[2]. Chiang CM, Roeder RG. Expression and purification of general transcription factors by FLAG epitope-tagging and peptide elution. Pept Res. 1993 Mar-Apr;6(2):62-4. PMID: 7683509.
[3]. Prickett KS, Amberg DC, Hopp TP. A calcium-dependent antibody for identification and purification of recombinant proteins. Biotechniques. 1989 Jun;7(6):580-9. PMID: 2698650.
[4]. Schuster M, Wasserbauer E, Ortner C, Graumann K, Jungbauer A, Hammerschmid F, Werner G. Short cut of protein purification by integration of cell-disrupture and affinity extraction. Bioseparation. 2000;9(2):59-67. doi: 10.1023/a:1008135913202. PMID: 10892539.
[5]. Hopp TP, Prickett KS, Price VL, et al. A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. Bio/technology (Nature Publishing Company). 1988;6:1204-1210. DOI: 10.1038/nbt1088-1204.
[6].Li Y. Commonly used tag combinations for tandem affinity purification. Biotechnol Appl Biochem. 2010 Feb 15;55(2):73-83. doi: 10.1042/BA20090273. PMID: 20156193.
FLAG tag Peptide 是一种包含肠激酶限制性位点的 8 肽 (Asp-Tyr-lys-Asp-Asp-Asp-ASP-ASP-Lys)。 FLAG Tag 肽融合系统用于免疫亲和层析，用于蛋白质鉴定和纯化。 FLAG 标签肽标签肽允许在非变性条件下洗脱[1,6]。 FLAG 标签肽可以融合到给定融合蛋白的 N 端或 C 端。
FLAG 标签肽标记肽融合系统包含一种独特且广泛有用的蛋白质鉴定和纯化技术。当共价连接到固体支持物上时，抗 Flag M1 抗体可用于在温和的钙依赖性亲和层析过程中快速纯化 FLAG 标签肽融合蛋白。 FLAG 标签肽融合蛋白通常从粗细胞匀浆或上清液开始一步纯化至均质，除了 pH 7.2 的生理盐水（含钙或 EDTA）外，从未将蛋白质暴露于其他条件。整个纯化过程可在数小时内完成[2-3]。
N-terminal FLAG tag Peptide融合蛋白可以通过琼脂糖亲和凝胶纯化[5]。一种快捷的蛋白质纯化程序，其中结合了机械细胞分解和通过将抗 Flag M1 抗体固定在磁性玻璃珠上进行的亲和纯化。将改性玻璃珠添加到酵母细胞中并在实验室涡旋混合器上混合。磁性玻璃珠捕获目标蛋白，同时破坏酵母细胞。通过与固定磁铁接触，磁性玻璃珠可以非常有效地与酵母细胞碎片分离。 EDTA 等螯合剂能够从磁性玻璃珠上洗脱 FLAG 标签肽融合蛋白[4]。