Part II: Currently Used Protein Labeling Strategies

Protein Quantum Dot Labeling

Principles: Quantum Dots is an inorganic nanocrystal that emits fluorescence at a specific wavelength according to its size. It has a very high extinction coefficient and its extinction coefficient is 10 to 100 higher than that of small molecule fluorophores and fluorescent proteins. The quantum yield of quantum dots is also good. Typical quantum dots contain a cadmium selenide (CdSe) or cadmium telluride (CdTe) core surrounded by a zinc sulfide (ZnS) shell. Quantum dots absorb a wide range of wavelengths from very short wavelengths to slightly below their emission wavelengths, so a single wavelength of excitation light allows quantum dots to multiply.

Application and features: Currently, water-soluble quantum dots and oil-soluble quantum dots can be coupled with molecules such as antibodies and streptavidin, and are used in biomarker detection, high-throughput coding, in vivo imaging, and dynamic tracing. However, the water-soluble quantum dots combined with biomacromolecules are bulky (about 10 nm to 30 nm in diameter), which hinders their passage through the cell membrane structure, so they can only be used for permeabilized cells, or only for extracellular proteins can be studied by proteins that are endocytosed by cells.

No.3 Other Labeling Strategies

Biotin labeling

Principle: Biotin is a useful tag for protein detection, purification and immobilization because it binds with high affinity to streptavidin (Avidin) and streptavidin (SA). The affinity of biotin-avidin is at least a million times greater than antigen-antibody binding, one of the strongest non-covalent interactions between proteins and ligands. In addition, biotin (MW = 244.3 Da) is much smaller than the enzyme label and therefore does not affect the natural function of the protein itself. Together, these features make the biotin-avidin strategy an ideal choice for many detection and fixation applications.

Biotinylated protein is a product of covalent binding of biotin to protein. Because biotinylated protein has the advantages of high affinity, high specificity and high sensitivity, it improves the efficiency of detection and purification of protein based on immunological methods.

Applications: The biotin-avidin system is widely used in flow cytometry, fluorescence imaging, Western blotting, and ELISA assays to increase signal output and higher sensitivity. A fluorescent conjugate of avidin or streptavidin is used to detect biotinylated biomolecules. Avidin or streptavidin enzyme conjugates are commonly used in Western blot, ELISA and in situ hybridization imaging applications. Avidin or streptavidin-conjugated magnetic beads and resins can be used to separate proteins, cells and DNA, as well as for immunoassays or bioscreening.

Enzyme Protein Conjugation

An enzyme marker typified by horseradish peroxidase (HRP) and alkaline phospholipase (AP) is suitable for coupling with a protein and preparing an antibody conjugate for immunodetection. These two markers are often used to label antibodies and used as an ELISA assay.

HRP is suitable for rapid color reaction. After adding substrate in ELISA, the OD450 value can reach 1.8~2.5 within 5~10 min, while AP is suitable for slow color reaction. The color development time is about 4~8 h. The yellow compound catalyzed is relatively stable and is suitable for quantitative detection of enzymatic kinetics.

About Author: MtoZ Biolabs is an international contract research organization (CRO) providing advanced proteomics, metabolomics, bioinformatics, and biopharmaceutical analysis services to researchers in biochemistry, biotechnology, and biopharmaceutical fields. It is specialized in quantitative multiplexed proteomics and metabolomics applications through the establishment of state-of-the-art mass spectrometry platforms, coupled with high-performance liquid chromatography technology.

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