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Protein labeling means that a marker is bound to the target protein in vivo or in vitro. The labeled protein remains its protein activity; and, the marker is used as either a tracer to indicate protein associated reactions, or an intervention agent that acts on the target protein or cells. With technological progress in recent years, especially emergence and application of green fluorescent protein (GFP), protein labeling plays an increasingly important role in the field of life sciences.
As an analytical detection approach to monitoring vital processes and detecting the target, protein labeling is also a means of intervention and treatment by virtue of protein activity and marker actions. For the past few years, recombination monoclonal antibodies are coupled with radioactive isotopes, immunotoxin or chemicals to prepare targeted drugs of precision treatment. This is a new method of serious disease treatment, including tumors and autoimmunity diseases.
According to types of markers, protein labeling can be classified into radioactive isotope labeling, fluorescein/fluorescent protein labeling, enzyme labeling, chemiluminescent substance (e.g., acridinium ester and luminol) labeling, and nano material (e.g., colloidal gold, electroselenium, leatexbeads and quantum dots) labeling, etc.
In accordance with protein labeling, corresponding technical solutions should be proposed according to protein features and marker types：
1.Radioactive isotope labeling: It is featured with high sensitivity, high specificity, high applicability and accurate positioning, etc.. Commonly used isotopes include 125I, 131I and 32P, etc.. The key to isotope labeling is found in conditions for radioactive intensity determination and appropriate specific activity. Corresponding labeling process includes experimental preparation, labeling experiment and radioactive waste treatment, etc.
2.Fluorescence labeling: Fluorescent materials are bound to the target protein to produce a fluorescent conjugate. In detail, fluorescent materials include fluorescein (e.g., FITC, rhodamine and 5-FAM), auto-fluorescent protein, and nanocrystalline materials (e.g., quantum dots). Based on different natures of various fluorescent materials, specific labeling processes may greatly vary.
3.Enzyme labeling: Common enzyme molecules used for labeling includes horse radish peroxidase, alkaline phosphatase and glucose oxidase. Corresponding coupling methods cover a glutaraldehyde reaction, a benzoquinone method and an azo method, etc.
4. Biotin labeling: Biotin with small molecular weight has a minor influence on protein activity. Importantly, both biotin and avidin are of high affinity, so that detection sensitivity can be substantially improved. Therefore, biotin labeling has become a commonly used effective protein labeling technique.
5. Other labeling methods: Through gene level manipulation, fluorescent protein genes are recombined with the target protein gene; then, the recombinant protein is expressed. Nano-particles can absorb protein by physical methods to produce a marker.
Now, multiple protein labeling techniques have been developed. On this basis, a convenient and stable bioinstrumentation process platform with high sensitivity and specificity. It can be applied in developing products for immunoassay, such as ELISA, immunochemiluminescence and nanogold/fluorescent microsphere immunochromatography. An enzyme labeling technique developed in our company is featured with a high labeling rate, high activity and high purity, etc.. Such a technique is applicable to labeling of protein molecules with diverse types. Furthermore, a fluorescent microsphere labeling technique with high sensitivity and a large linearity range has been also developed. Labeling microspheres are of uniform granularity, good monodispersity and high luminous efficiency. Such a technique can be extensively applied in high-throughput immunoassay and analysis as well as gene detection, etc.
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