Product Introduction
1.1 BioMag series
The "Bio" in the BioMag series is derived from the prefix of our company name "Biodragon", and "Mag" stands for "Magnetic". All products containing BioMag are polymer magnetic microsphere products. At present, the BioMag series is mainly based on antibody affinity method labeled protein IP kits. It includes six types of tag IP kits: Anti-His-tag, Anti-Myc-tag, Anti-HA-tag, Anti-Flag-tag, Anti-GFP-tag and Anti-RFP-tag. In the future, we will develop more BioMag polymer magnetic bead-related products based on customer demands.
1.2 Flag Tag
The Flag peptide is one of the most commonly used tags in the expression and purification process of fusion proteins. It can be attached to the N-terminal, C-terminal or the middle of the sequence of the fusion protein, has excellent hydrophilicity, and is more likely to be displayed on the surface of the fusion protein, with a smaller possibility of interfering with the function of the fusion protein. This tag also has an advantage: when attached to the N-terminal of the target protein, it can be excised from the lysine position at the C-terminal of the sequence by Enterokinase (EK protease) without leaving redundant residues.
1.3 Anti-Flag antibody
The Flag tag peptide is a hydrophilic short peptide composed of 8 amino acids (DYKDDDDK). The DYKDDDDK peptide segment is also often referred to as the Flag peptide segment. A series of antibodies against DYKDDDDK tags include Anti-Flag M1, Anti-Flag M2, Anti-Flag M5, etc. Among them, M2 antibody is widely used in immunohybridization, immunoprecipitation, affinity purification and other directions. The Anti-Flag antibody of our company has a high affinity and is suitable for protein-protein interaction experiments such as WB and IP.
1.4 Components of the Kit
Antibodies have specific binding to the corresponding tags. By conjugating antibodies onto Beads, the fusion proteins with the corresponding tags can be bound to the Beads. After steps such as removing impurity proteins with buffer solution, the fusion protein can be eluted from the Beads using denaturants or high-temperature treatment, etc. For ease of use, this kit provides a variety of reagents to facilitate customers' better conduct protein interaction experiments.
1.5 Description of Elution buffer
Elution Buffer D contains a high concentration of urea and detergent, which can fully elute the tag protein and ensure that only a small amount of antibodies fall off the magnetic beads. However, there may still be a small amount of target protein remaining on the magnetic beads. It can also be directly added to the SDS Loading Buffer in the kit and treated at a high temperature of 95-100℃ for 5-10 minutes. The target protein can be completely detached from the magnetic beads, but the antibody will also completely fall off. When conducting subsequent WB experiments, the species of the secondary antibody should be carefully selected.
1.6 Positive control protein
The kit provides a red fluorescent protein with a Flag tag. This protein emits visible red light under natural light and can be used to verify the binding capacity of Beads and eliminate possible deviations in the operation process. This protein is eukaryotic expressed and is shown as three bands with different molecular weights on SDS-PAGE. The binding time, binding conditions and elution conditions of different proteins with Flag tags to Beads are similar and can be referred to in parallel. However, considering that the binding of proteins to Beads is concentration-dependent, the operating conditions for positive control proteins should not be blindly followed without change.
1.7 Properties of Magnetic Beads
Polymer magnetic beads are made by using high-molecular polymerization technology to perfectly combine superparamagnetic materials and high-molecular materials. They have a faster magnetic response while maintaining the good dispersion of microspheres, extremely low non-specific adsorption and more abundant binding sites. BioMag Anti-Flag Beads are prepared based on the principle of high affinity between Biotin and streptavidin (SA) proteins. Firstly, SA proteins are densely coupled to BioMag magnetic beads, and then biotinylated Flag antibodies are non-covalently coupled to SA polymer magnetic beads. Thus, it avoids the inactivation of antibodies caused by chemical covalent coupling. Therefore, BioMag Anti-Flag Beads have a higher antibody coupling density and antibody specific activity. It can be used for protein interaction experiments such as IP and Co-IP of Flag tag proteins secreted and expressed in prokaryotic and eukaryotic cell lysates or culture supernatants. Magnetic beads can be separated through magnetic racks or automatic separation instruments. The method is simple, efficient and convenient.