Part I: Currently Used Protein Labeling Strategies

No.1 Metabolic Labeling

Metabolic labeling strategy is an in vivo labeling method in which cells are "fed" with chemically labeled nutrients that are then incorporated into newly synthesized proteins, nucleic acids or metabolites. We can then collect the cells and isolate these molecules to obtain a global view of the cellular biological processes.

Protein Isotope Labeling

Principle: Protein isotope labeling is a classic protein tracing and proteomic quantification technique that replaces the corresponding amino acids in the cell culture medium with essential amino acids labeled with natural isotopes (light) or stable isotopes (heavy), so that newly synthesized proteins can labeling is carried out by incorporation of amino acids containing different isotopes during cell growth.

Application example: SILAC (Stable Isotope Labeling with Amino acids in Cell Culture), a stable isotope labeling of amino acids in cell cultureis, is a popular metabolic labeling method for proteomics research. In combination with mass spectrometry, SILAC quantifies two cultures or cells by labeling one of the cultures or cell lines with a heavy amino acid (eg, 15N- or 13C-lysine) and adding normal light amino acids to another group. The difference in protein abundance between lines. The lysed proteins of the cells grown under these two conditions are then mixed in equal proportions of the number of cells or the amount of protein, separated and purified, and then identified by mass spectrometry, and compared according to the area comparison of the two isotopic peptides in the first-order mass spectrum. Quantitatively, a relative assessment of protein abundance differences under both conditions was obtained.

Other Protein Metabolism Markers

If mass spectrometry conditions are not available, a bio-orthogonal reaction based metabolic labeling method can be used. In bioorthogonal systems, cells are "fed" with molecular building blocks labeled with chemical groups that are unreactive with natural biological reactive groups. The exogenous compound added to the reaction partner containing the group initiates a chemical reaction to couple the labeled biomolecule to the desired functional group.

Application example: The cells are "fed" with a structural unit containing an azide group and then treated with a reagent containing a phosphine group after the experiment. The azide and phosphine groups are coupled by a Staudinger ligation reaction, azide and Phosphine groups are generally not present in biological systems and therefore these molecules are inert.

No.2 Fluorescent Labeling Strategy

Materials including autofluorescence proteins, small molecule fluorescent marker dyes, nanocrystalline materials (ie, quantum dot materials), small molecular protein labels can be used as fluorescent markers. These markers can be used to study the expression of target proteins, localization in cells, interaction, activity status and other indicators combined with fluorescence imaging detection instruments.

Fluorescent Protein Label

If the target protein is fused to a fluorescent protein, intracellular observation of the target protein can be achieved, which can be used for multicolor labeling and fluorescence resonance energy transfer (FRET) applications for visualizing translocation of proteins and other subcellular structures, studying proteins-protein colocalization, detecting the onset of gene expression from different promoters and the separation of mixed cell populations. From the early development of green fluorescent protein GFP to the present, we already have fluorescent proteins covering a variety of spectral regions, including green fluorescent protein, blue and blue-green fluorescent protein, yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, different Spectral fluorescent proteins differ in brightness, photostability, and molecular size.

Fluorescent Dye Protein In Vitro Labeling

Fluorescent dye-labeled protein or peptide technology is a common protein in vitro labeling technology. In addition to GFP and other fusion-expressing fluorescent proteins, the target protein can be directly labeled for downstream experiments such as live tracing and cell sorting by fluorescent dye labeling.

Principle: The compound on which the fluorescent label dependent is called fluorescent substance. A fluorescent substance refers to a compound having a chemical structure of a conjugated double bond system, and when excited by ultraviolet light or blue-violet light. It can be excited to become an excited state, and when it is restored from the excited state, it emits fluorescence. Protein fluorescent labeling technology uses a fluorescent substance to covalently bind to a certain group of a target molecule, and uses its fluorescent properties to provide information on the object to be studied.

Application and features: Activated fluorescent dyes such as fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (AMC), Rhodamine B or Alexa Fluor dyes, etc., can be used to label antibodies or proteins A functional group that generates a molecular probe and is detected by fluorescence imaging. When chemically labeling specific antibodies or other purified biomolecules with fluorescent dyes, they become fluorescent probes for detecting target antigens or interacting partners for cell imaging, flow cytometry, Western blotting, and enzyme-linked immunosorbent assays. Adsorption experiment (ELISA). Because fluorescent markers have the advantages of no radioactive contamination and easy operation, they are widely used in many research fields such as protein function research and drug screening.