The role of NHS group in acridinium ester
Some acridine esters or acridine sulfonamides contain NHS groups. This group does not participate in the chemiluminescence reaction, but it is a key group for acridine esters to directly label and cross-link proteins, antibodies or enzymes. Amine-reactive crosslinkers are widely used in acridinium ester, fluorescein and other biomolecular probes.
Acridinium NHS Ester Marked Protein
The structure of NHS ester in acridinium ester
NHS ester refers to the special ester (- COO-N-). This ester can react with the primary amine (primary amino group) on the protein. The CO bond connected to the carbonyl group in the NHS ester is broken to remove the NHS group, and then the carbonyl group is connected to the protein amino group to form a -CONH-amide bond (that is, the carboxyamino group in the protein). Peptide bond formed by dehydration condensation), so as to connect with the protein to achieve the purpose of labeling the protein. Follow-up chemiluminescence reaction to detect the acridinium-labeled protein.
Advantages of NHS esters
There are many chemical groups that react with primary amines (-NH2), and some acridinium esters are used to label proteins or peptides in other ways, but NHS esters are more versatile and common. Primary amines exist in the N-terminus of each polypeptide chain or protein and the side chain of lysine (Lys, K) amino acid residues, that is, most proteins can be labeled with NHS esters. These primary amines are positively charged at physiological pH; therefore, they mainly occur on the outer surface of the tertiary structure of natural proteins and easily react with the binding reagents introduced into the aqueous medium. In addition, among the chemical functional groups of typical biological or protein samples, primary amines are especially nucleophilic, and are easy to target with several reactive groups, such as NHS esters, isothiocyanates and some acridines. Carbodiimide used in esters.
Attention to the use of acridinium ester NHS ester
NHS ester hydrolysis competes with the reaction of primary amines. The rate of hydrolysis increases with the increase of pH, so the low-concentration protein recording rate will decrease. Therefore, when labeling proteins with acridine esters, it is preferred to complete under physiological to weakly alkaline pH (7.2-9). . The buffer can be phosphate, carbonate, HEPES or borate buffer. Tris buffer containing primary amines will compete and be incompatible. At the end of the labeling step, it can be quenched with Tris or glycine buffer. Desheng is a manufacturer of chemiluminescence reagents, which can provide acridinium esters and related buffers containing NHS and other cross-linked structures.
In chemiluminescence analysis, the luminescence intensity of acridine ester is influenced by various factors, such as reaction medium, temperature, time, and excitation light source energy. To achieve good detection results, it is necessary to comprehensively consider and optimize these factors. Meanwhile, attention should be paid to controlling and standardizing experimental conditions to ensure accurate and reliable results. Thoroughly studying these influencing factors will help promote the development of chemiluminescence analysis methods.