FLAG 和 3xFLAG 标签：
A Proven System for Detection and Purification of Proteins The FLAG Expression System is an established way to express, purify and detect recombinant fusion proteins. FLAG and 3xFLAG have proven utility in numerous applications such as Western blotting, immunocytochemistry, immunoprecipitation, flow cytometry, protein purification, and in the study of protein-protein interactions, cell ultrastructure and protein localization. These small hydrophilic tags facilitate superior detection and purification of recombinant fusion proteins when using our highly specific and sensitive ANTI-FLAG antibodies. Ideal Epitope Tags: Small, Hydrophilic and Cleavable The FLAG expression system utilizes a short, hydrophilic 8-amino acid peptide that is fused to the recombinant protein of interest. Because of its hydrophilic nature, the FLAG peptide is likely to be located on the surface of the fusion protein. As a result, the FLAG peptide is easily accessible for cleavage by enterokinase (Ek) and for detection with antibodies. In addition, because of the small size of the FLAG peptide tag, it is not likely to obscure other epitopes, domains, or alter function, secretion, or transport of the fusion protein. The 3xFLAG system is an improvement upon the original system by fusing 3 tandem FLAG epitopes (22 amino acids). Detection of fusion proteins containing 3xFLAG is enhanced up to 200 times more than any other system. Like the original FLAG tag, 3xFLAG is hydrophilic, contains an Ek cleavage site, and is relatively small. Therefore, the risk of altering protein function, blocking other epitopes or decreasing solubility is minimized.
3xFLAG System Expression Vectors for Ultra-Sensitive
重组蛋白的检测
The 3xFLAG system is an improvement upon the original system by fusing 3 tandem FLAG epitopes for a total of 22 amino acids. Detection of fusion proteins containing 3xFLAG is enhanced up to 200 times more than any other system. Like the original FLAG tag, 3xFLAG is hydrophilic, contains an Enterokinase cleavage site, and is relatively small. Therefore, the risk of altering protein function, blocking other epitopes or decreasing solubility is minimized. In cases of low-level expression, 3xFLAG is ideal. Our selection of CMV vectors provides options for transient or stable expression of 3xFLAG fusion proteins.
Ultrasensitive System - Detect < 10 femtomoles using ANTI-FLAG Antibodies; Ideal for cases of low-level expression in mammalian cells.
Superior Detection - 20-200x more sensitive than any other system.
Versatile - Enhanced detection for immunoprecipitation, Western blots and immunocytochemistry.
Wide Selection of Detection and Purification Products - ANTI-FLAG antibodies, resins, and affinity capture plates.
CMV启动子：
Our mammalian expression vectors contain the strong CMV promoter for high-level constitutive expression in mammalian cells. The strong human cytomegalovirus (CMV) promoter regulatory region drives constitutive protein expression levels as high as 1 mg/L in COS cells. For less potent cell lines, protein levels are typically ~0.1 mg/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication permissive COS cells. Vectors containing the preprotrypsin leader (PPT) sequence direct secretion of FLAG fusion proteins into the culture medium for purification using ANTI-FLAG antibodies, resins, and plates. 载体介绍： The p3XFLAG-CMV-9 Expression Vector is a 6.4 kb derivative of pCMV5 used to establish transient or stable expression of secreted N-terminal 3XFLAG fusion proteins in mammalian cells. The vector encodes three adjacent FLAG epitopes (Asp-Tyr-Lys-Xaa-Xaa- Asp) upstream of the multiple cloning region. This results in increased detection sensitivity using ANTIFLAG M2 antibody. The third epitope includes the enterokinase recognition sequence, allowing cleavage of the 3XFLAG peptide from the purified fusion protein.
p3XFLAG-CMV-9 expression vector is a shuttle vector for E. coli and mammalian cells. Efficiency of replication and genomic integration is optimal when using an SV40 T antigen expressing host, such as COS cells. A related vector, p3XFLAG-CMV-3, has been used for stable transfection of HEK 293 cells. Efficiency of replication and genomic integration is optimal when using an SV40 T antigen-expressing host.
The p3XFLAG-CMV-7-BAP Control Plasmid is a 6.2 kb derivative of pCMV5 used for transient intracellular expression of N-terminal 3XFLAG bacterial alkaline phosphatase fusion protein in mammalian cells. The vector encodes three adjacent FLAG epitopes (Asp-Tyr-Lys-Xaa-Xaa-Asp) upstream of the multiple cloning region. This results in increased detection sensitivity using ANTI-FLAG M2 antibody. The third FLAG epitope includes the enterokinase recognition sequence, allowing cleavage of the 3XFLAG peptide from the purified fusion protein.
The promoter-regulatory region of the human cytomegalovirus drives transcription of FLAG-fusion constructs. The preprotrypsin leader sequence precedes the FLAG sequence. The aminoglycoside phosphotransferase II gene (Neo-r) confers resistance to aminoglycosides such as G418 allowing for selection of stable transfectants.
