Fluorescent protein labeling molecular are typically sold wi

Fluorescent protein labeling molecular are typically sold with one of two binding groups: 1) NHS ester and 2) Maleimide. What are the differences? Why not just sell one?

Solution

Answer:

NHS (N-HydroxySuccinimide) ester is a reactive compounds suitable for the modification of amino groups.

NHS is most common type of activated esters for cross linking and labelling proteins such as antibodies involve the use of chemical group that react with primary amine (-NH2). Primary amines exists at the N-terminal of polypeptide chain and in the side chain of lysine. These primary amines are positively charged at physiological pH, therefore they occur primarily on the outer surface of protein surface where they are readily accessible to conjugation reagents introduced into the aqueous medium.

Sulfhydryl-reactive chemical groups in biomolecular probes for labeling and crosslinking cysteines and other sulfhydryls include maleimides.

Sulfhydryls, also called thiols, exist in proteins in the side-chain of cysteine (Cys, C) amino acids. Pairs of cysteine sulfhydryl groups are often linked by disulfide bonds (–S–S–) within or between polypeptide chains as the basis of native tertiary or quaternary protein structure. Typically, only free or reduced sulfhydryl groups (–SH) are available for reaction with thiol-reactive compounds.

Sulfhydryl groups are useful targets for protein conjugation and labeling. First, sulfhydryls are present in most proteins but are not as numerous as primary amines; thus, crosslinking via sulfhydryl groups is more selective and precise. Second, sulfhydryl groups in proteins are often involved in disulfide bonds, so crosslinking at these sites typically does not significantly modify the underlying protein structure or block binding sites. Third, the number of available (i.e., free) sulfhydryl groups can be easily controlled or modified.

So, they are not sold singly due to their different labelling targets, one of which labels primary amines while the other labels thiols.