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3XFLAG Peptide is an important peptide marker sequence widely used in protein expression and purification processes in biomedical research. Molecular formula C120H169N31O49S, CAS 402750-12-3. Composed of 23 amino acids, its sequence contains three repeated Asp Tyr Lys Xaa Xaa Asp motifs, which give it specific structural features. Has good solubility in specific solvents. For example, in DMSO, its solubility is high, making it convenient for operations and experiments in solution. In addition, it can also achieve good dissolution effect in water through mild heating. It also has some special properties. For example, it has good antigenicity and reactivity, and can bind to specific anti FLAG antibodies. This binding ability makes it widely applicable in experiments such as immunoprecipitation. By competitively binding to Anti FLAG antibodies, the FLAG fusion expressed protein that binds to Anti FLAG antibodies can be eluted during immunoprecipitation, thereby achieving localization, detection, and purification of the target protein.
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Customized Bottle Caps And Corks:
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Chemical Formula |
C120H169N31O49S |
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Exact Mass |
2860 |
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Molecular Weight |
2862 |
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m/z |
2861 (100.0%), 2860 (77.0%), 2862 (32.9%), 2862 (31.4%), 2863 (22.1%), 2862 (11.5%), 2863 (10.1%), 2861 (8.8%), 2864 (8.1%), 2862 (7.8%), 2863 (7.4%), 2863 (5.3%), 2863 (4.5%), 2862 (3.5%), 2864 (3.3%), 2864 (3.2%), 2865 (2.2%), 2865 (2.1%), 2864 (2.1%), 2862 (1.9%), 2862 (1.9%), 2861 (1.5%), 2864 (1.5%), 2861 (1.4%), 2864 (1.4%), 2863 (1.2%), 2864 (1.2%) |
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Elemental Analysis |
C, 50.36; H, 5.95; N, 15.17; O, 27.39; S, 1.12 |

3XFLAG Peptide is a tag used in protein research, consisting of 8 amino acids with the sequence DYKDHDGDYKDHDIDYKDDDDK. Its main function is to serve as a marker for protein expression and purification, providing a convenient and efficient means in research.

1. Affinity purification:
It can be used as a label for protein purification, and through its high affinity with anti FLAG antibodies, the target protein can be easily purified. Usually, the target protein is co transfected into cells with an expression vector containing 3X FLAG PEPTIDE, and then purified by affinity using anti FLAG antibodies to quickly and efficiently obtain a high purity target protein.
2. Immunological testing:
Due to its high immunogenicity, it can be used to detect protein expression and localization. By utilizing specific anti FLAG antibodies, experimental techniques such as immunoblotting and immunofluorescence staining can be performed to quickly and accurately detect the expression level and distribution of target proteins.
3. Clone filtering:
It can also be used for the convenience of protein clone screening. In the construction process of protein expression vectors, connecting the gene sequence of the target protein with the 3XFLAG Peptide sequence can facilitate the screening of positive clones with the target protein through immune detection and other methods, thereby accelerating the process of protein function research.
4. Immunoprecipitation:
It can also be used for protein immunoprecipitation experiments. By binding proteins containing 3X FLAG PEPTIDE tags to anti FLAG antibodies, and then using adsorption materials such as protein A/G agarose or magnetic beads, the target protein and its interacting partners can be selectively precipitated from cell extracts to study the protein interaction network.


5. Drug screening:
It can also be used in drug screening research. By combining the 3X FLAG PEPTIDE tag with the protein of interest, high-throughput screening of the protein can be achieved. This screening method can be used to discover new drug targets, evaluate drug protein interactions, and so on.
6. Protein function research:
The introduction of labels does not significantly affect the natural structure and function of proteins, and can therefore be used to study the biological functions, subcellular localization, interactions, and signal transduction of proteins. By introducing the 3X FLAG PEPTIDE tag, in-depth research can be conducted on the biological characteristics of proteins, which helps to reveal their mechanisms of action in cellular biology and physiology.

3XFLAG Peptide is a peptide containing 8 amino acids, with the sequence DYKDHDGDYKDHDIDYKDDDDK. It is commonly used as a protein tag for convenient purification, detection, and research of target proteins. In the process of biosynthesis, it can be prepared through chemical synthesis or genetic engineering methods. The following are the detailed steps of genetic engineering methods:
1. Design and synthesize genes:
Firstly, it is necessary to design an artificial gene containing the 3X FLAG PEPTIDE sequence. This gene should contain the 3X FLAG PEPTIDE sequence, as well as appropriate promoters, terminators, and selection markers (such as antibiotic tolerance genes). The design of genes should consider the characteristics and expression needs of the target host.
2. Gene cloning:
Next, the designed artificial genes will be cloned into appropriate expression vectors. Commonly used vectors include plasmids, viral vectors, etc. The cloning process typically includes the following steps:
-PCR amplification: Using polymerase chain reaction (PCR) to amplify the target gene sequence from template DNA.
-Digestion and connection: PCR products are digested with expression vectors using restriction endonucleases, and then connected together by DNA ligases.
-Transformation: Transforming the connected expression vector into bacterial hosts such as Escherichia coli to form a recombinant plasmid.
-Identification: Recombinant plasmids were identified through PCR, enzyme digestion, DNA sequencing, and other methods to confirm that the target gene has been successfully cloned into the vector.
3. Expression target protein:
Transfect the constructed expression vector into target host cells, such as mammalian cells, yeast cells, insect cells, etc. Within cells, the expression vector is transcribed into mRNA and then translated into proteins. 3X FLAG PEPTIDE will be expressed together with the target protein as a protein tag.
4. Protein extraction:
Collect transfected cells and perform cell fragmentation, centrifugation, and other treatments to obtain the total protein extract.
5. Purified protein:
Using affinity purification techniques such as immunoprecipitation and affinity chromatography, target proteins containing 3X FLAG PEPTIDE tags were purified from the total extract. This usually involves binding anti FLAG antibodies to proteins containing 3X FLAG PEPTIDE, and then separating them using affinity chromatography columns or magnetic beads.
6. Identify proteins:
The purified protein was identified and quantified by SDS-PAGE gel electrophoresis, immunoblotting, mass spectrometry and other technologies to ensure its purity and integrity.
Adverse reactions
In biomedical research and protein related experiments, tag peptides play a crucial role. They can assist researchers in purifying, detecting, and locating target proteins, greatly promoting the development of protein science. 3XFLAG Peptide is a common and widely used tag peptide composed of three repeated FLAG tag sequences. The FLAG tag was originally designed by Hopp et al. in 1988, and later developed into variants such as 3XFLAG, which have high affinity and specificity and can bind to specific antibodies, facilitating subsequent isolation and identification. Although 3XFLAG Peptide has brought many conveniences in experiments, like other biological agents, it may also cause some adverse reactions.
Immune related adverse reactions
Immunogenicity
Immunogenicity refers to the ability of a substance to stimulate the body's immune system to produce an immune response. 3XFLAG Peptide, as an exogenous peptide segment, may be recognized by the body's immune system as a "non self" component, thereby triggering an immune response. Although the 3XFLAG sequence is relatively short, in some cases it may still activate B cells and T cells, leading to the production of antibodies. These antibodies not only bind to the free 3XFLAG Peptide, but may also recognize and bind to fusion proteins expressing the 3XFLAG tag, affecting the normal function and distribution of the fusion protein.
Allergic reactions
Allergic reaction is an abnormal immune response caused by the immune system being overly sensitive to certain allergens. Individual individuals may experience allergic reactions when using 3XFLAG Peptide for experimentation or treatment. The severity of symptoms of allergic reactions varies. Mild cases may present with local symptoms such as itching, rash, and erythema, while severe cases may trigger systemic allergic reactions such as anaphylactic shock, characterized by a sharp drop in blood pressure, difficulty breathing, and loss of consciousness, posing a serious threat to life safety.
Allergic reactions
The mechanism of allergic reactions is mainly related to IgE antibodies in the immune system. When the body first comes into contact with 3XFLAG Peptide, the immune system recognizes it as an allergen and stimulates B cells to produce specific IgE antibodies. These IgE antibodies attach to the surface of mast cells and eosinophils. When the body comes into contact with 3XFLAG Peptide again, it binds to IgE antibodies on the cell surface, triggering mast cells and eosinophils to release bioactive mediators such as histamine and leukotrienes, leading to the occurrence of allergic symptoms.
Risk of autoimmune diseases
Long term or repeated exposure to 3XFLAG Peptide may increase the risk of developing autoimmune diseases. Autoimmune diseases refer to a type of disease in which the body's immune system mistakenly attacks its own tissues and organs, resulting in tissue damage and functional impairment. 3XFLAG Peptide may induce the immune system to produce cross reactive antibodies and T cells through a molecular simulation mechanism, where its amino acid sequence is similar to the sequence of certain proteins in the body itself. These immune cells and antibodies not only attack the foreign 3XFLAG Peptide, but also inadvertently damage the body's normal tissues, leading to autoimmune diseases. For example, certain autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis may be related to autoimmune reactions induced by exogenous substances. Although there is currently no clear evidence to suggest that 3XFLAG Peptide directly causes autoimmune diseases, this potential risk cannot be ignored.
The impact on cellular function
Cytotoxicity
In some experimental studies, it has been found that high concentrations of 3XFLAG Peptide may have toxic effects on cells. The manifestations of cytotoxicity include changes in cell morphology, disruption of cell membrane integrity, alterations in intracellular enzyme activity, and inhibition of cell proliferation. Its mechanism of action may be related to the interaction between 3XFLAG Peptide and the cell membrane. Due to the strong negative charge of 3XFLAG Peptide, it may bind to positively charged components on the cell membrane through electrostatic interactions, altering the permeability and fluidity of the cell membrane, leading to leakage of intracellular substances or entry of harmful substances from the outside, thereby affecting the normal physiological functions of the cell. In addition, 3XFLAG Peptide may also enter the interior of cells, interfere with signaling pathways, protein synthesis, and other processes, further exacerbating cell damage.
Effects on cellular signaling pathways
3XFLAG Peptide may interfere with intracellular signaling pathways and affect normal physiological activities of cells. The cellular signaling pathway is a collective term for a series of molecular events in which cells respond to external stimuli, regulating important processes such as cell growth, differentiation, and apoptosis. 3XFLAG Peptide may interfere with the normal transmission of signaling pathways by interacting with key molecules in the signaling pathway, altering the activity or expression levels of signaling molecules. For example, it may bind to receptors on the cell surface, competitively inhibiting the binding of natural ligands to receptors and blocking signal transmission; Alternatively, it can enter the cell and bind to signal transduction proteins, affecting protein phosphorylation, dephosphorylation and other modification processes, interfering with signal transmission and amplification. This interference with cellular signaling pathways may lead to cellular dysfunction, affecting processes such as cell proliferation, differentiation, and apoptosis, which in turn can have adverse effects on the normal development and function of tissues and organs.
Effects on cellular metabolism
Cellular metabolism is the general term for a series of chemical reactions that occur within cells, providing energy and material basis for cellular life activities. 3XFLAG Peptide may affect cellular metabolic processes, interfere with cellular energy production and substance synthesis. For example, it may affect the function of mitochondria, which are the main sites for aerobic respiration and energy production in cells. 3XFLAG Peptide may alter mitochondrial membrane permeability and potential by interacting with proteins or lipids on the membrane, affecting mitochondrial respiratory chain function and ATP synthesis, leading to insufficient cellular energy supply. In addition, 3XFLAG Peptide may also interfere with intracellular processes such as glucose metabolism and lipid metabolism, affecting the balance of substance synthesis and decomposition in cells, and having a negative impact on normal physiological functions of cells.
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