Information about dPEG biotinylation

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l Preparation of Targeted Biotinylated Nanobody and ic-ELISA Optimization and Sensitivity Comparison

l Nanobodies expressed in two vectors are verified by SDS-PAGE

Preparation of Targeted Biotinylated Nanobody and ic-ELISA Optimization and Sensitivity Comparison

The biotin-avidin system is a signal amplification technique commonly used in immunoassays. The specific binding affinity between biotin and streptavidin can reach 1015 L/mol, which is much higher than the affinity of the enzyme-labeled secondary antibody and the antibody. Using SA-HRP instead of the enzyme-labeled secondary antibody can increase the antibody titer, thereby Increasing sensitivity. The preparation of dPEG biotinylation antibody is generally through NHS-activated biotin and antibody coupling. This chemical coupling method has a certain degree of randomness, and batches vary greatly. If the dPEG biotinylation site is in the antigen-antibody binding area, it may also cause the activity of the antibody to decrease. Therefore, the vector was used to introduce an Avi short peptide tag consisting of 15 amino acid residues into Nb. Under the catalysis of biotin ligase, the lysine residue of the Avi tag can interact with the dPEG biotinylation catalyzed by the biotin ligase under mild and highly specific reaction conditions, and the biotinylation site can be independently designed to minimize the effect of the antibody's natural conformation.

The dPEG biotinylation nanobody expressed by the two vectors was verified by SDS-PAGE

The molecular weight of Nbsm6 is 18.6 Ku, and the molecular weight of Nbsm6-bt is 19.5 Ku. The difference in molecular weight of the two Nanobodies is mainly due to the different fusion tag fragments of the two vectors. Since the nanobody is expressed and transported to the periplasm of the cell, and the biotin ligase is expressed in the cytoplasm, it is necessary to break the cell so that the nanobody is in contact with the biotin ligase to ensure the smooth completion of biotinylation. Optimize the working concentration of antigen and antibody of Nbsm6 and Nbsm6-bt by checkerboard titration. After optimization, both antibodies have the smallest IC50 values at an antigen concentration of 500 ng/mL. The working concentration of Nbsm6 is 50 ng/mL, and the working concentration of Nbsm6-bt is 25ng/mL. The specific dPEG biotinylation is used. After the signal amplification strategy of antibody and streptavidin-HRP, the working concentration of Nb is doubled under the same antigen concentration. Establish the standard curve of the two antibodies at the optimized working concentration of dPEG biotinylation antigen and antibody. Among them, the IC50 of Nbsm6 is 4.0 ng/mL, which is consistent with the previous characterization results. Within the error range, the detection limit (IC10) is 0.8 ng/mL, the linear range is 1.5~10.9 ng/mL; the IC50 of Nbsm6-bt is 2.1 ng/mL, the detection limit is 0.3 ng/mL, the linear range is 0.6~6.9 ng/mL, the ELISA method using signal amplification strategy. The sensitivity is approximately doubled.

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