LabelingIn-Depth Guide to Fluorescent Labeling Principles and Influencing Factors

When detecting specific indicators, pre-labeled Flow Cytometry Antibodies may not always be available, so you may need to label antibodies yourself before exploring their applications. Today, we will explain the principles of fluorescent labeling and the factors that affect it, giving you a better understanding of these experiments to make your work more effective and efficient.

I. Principles of Fluorescent Labeling

Antibodies are proteins made from amino acids through a process called dehydration condensation. Although there are hundreds of amino acids in nature, only about 20 are used to build proteins in living organisms. Amino acids are organic compounds with both basic amino groups and acidic carboxyl groups, and most naturally occurring amino acids contain primary amines. During dehydration condensation, the carboxyl group (-COOH) of one amino acid bonds with the amino group (-NH2) of another, releasing a molecule of water. This process forms peptide bonds (amide bonds) between the amino acids. After dehydration condensation, only the primary amino groups of lysine residues and the N-terminal amino acids of proteins are retained.

Figure 1. Schematic of amino acid dehydration condensation

Fluorochromes, which have conjugated double bond systems in their chemical structure. These fluorochromes can be excited by specific light and emit fluorescence when they return to their ground state. By covalently bonding or physically adsorbing fluorophores onto a specific group of the target molecule, we can utilize their fluorescent properties to gather information about the molecule.

Certain small molecule fluorophores or biotin can be activated and specifically react with primary amines on proteins or peptides within a certain pH and temperature range, forming stable amide bonds and thereby labeling the proteins or antibodies. Additionally, some fluorophores, once activated, can react specifically with intrinsic or introduced chemical reactive groups on proteins or antibodies under certain conditions, forming stable chemical bonds and thereby coupling the label to the proteins or antibodies.

Figure 2. Schematic of fluorophore molecules bonding with proteins or antibodies

In summary, the principle of fluorescent labeling is that activated fluorophores react with reactive groups on proteins or antibodies under specific conditions, thereby coupling the label to the proteins or antibodies. This technology is advantageous due to its non-radioactive nature and simple operation, leading to its widespread application.

II. Factors Affecting Fluorescent Labeling

Interference from Inorganic Salt Components

Components within proteins that interfere with labeling, such as glycine, imidazole, Tris, sodium azide, thimerosal, Proclin, etc., can affect the final labeling outcome. If the protein sample to be labeled contains such components, they should be removed by dialysis, desalting, or multiple ultrafiltration steps before labeling.

Carrier Protein Interference

Initial proteins containing carrier proteins like BSA or gelatin, and some protein preservation processes also involve adding BSA as a stabilizer. These carriers can bind with fluorophores, thus affecting the binding of dye molecules to the target protein during labeling. If the protein sample to be labeled contains carrier proteins, use affinity purification or other chromatographic methods to remove these interfering components, or use BSA removal kits (Cat. No.: E-LK-R002) and accurately quantify protein concentration before labeling.

Improper Experimental Operation

Incomplete mixing of dye and protein, or other operational errors, can affect the final fluorescent labeling result. Strictly follow the protocol instructions during labeling.

Inaccurate Initial Protein Concentration

Control the protein concentration before labeling and calculate the dye usage amount accordingly. If the initial protein concentration is inaccurate, it can lead to under-labeling or over-labeling, affecting subsequent experimental results.

Improper Dye Storage

Some dyes are prone to moisture-induced hydrolysis and inactivation. Store them with desiccants at -20°C or -80°C. To prevent condensation, bring the dye to room temperature before use.

III. Elabscience® Related Labeling Products (Partial)

Fluorescent Dyes

Labeling Kits