A Monoclonal Antibody That Discriminates Between SNAP-Tagged and CLIP-Tagged Proteins

Abstract

SNAP-tag technology allows recombinant proteins to be covalently labeled to O(6)-benzylguanine (BG)-modified substrates with 1:1 stoichiometry. By attaching according fluorophores, this method is ideally suited for in vitro and in vivo imaging, as well as protein interaction analyses. Fluorophores modified with BG react with the SNAP-tag, whereas those modified with O(2)-benzylcytosine (BC) conjugate to a more recent derivative known as the CLIP-tag. The orthogonal substrate specificity of the SNAP- and CLIP-tags extends the range of applications by allowing double labeling. We previously developed a monoclonal antibody (mAb) that recognizes both tags. In this study, we describe a new mAb, which is specific for the SNAP-tag alone. Therefore, this mAb allows discrimination between SNAP- and CLIP-tags within a broad range of immunological methods, including enzyme-linked immunosorbent assays, western blotting, flow cytometry, and immunohistochemistry.

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“…In summary, these mutations led to the generation of 'suicidal enzymes' such as CLIP-tag which can react specifically and rapidly with O 2 -benzylcytosine derivatives (BC-derivatives) and form an irreversible covalent bond between BC-ligands and cysteine residues within the CLIP-tag active site of the fusion protein. Therefore, CLIP-tag can be used as a selflabeling conjugation method for visualization of fusion proteins in living cells, as well as for enzyme-linked immunosorbent assays, western blotting, flow cytometry and immunohistochemistry [101,102]. SNAP-tag is a self-labeling enzyme, resulting from an engineered version of the 20 kDa human DNA repair protein AGT that specifically and rapidly reacts with BG derivatives.…”

“…SNAP-tag is a self-labeling enzyme, resulting from an engineered version of the 20 kDa human DNA repair protein AGT that specifically and rapidly reacts with BG derivatives. SNAP-tag has a 50-fold increased reactivity with BG-modified compounds when compared to AGT, which under normal conditions functions to remove alkyl adducts from the O 6 and the O 4 positions of guanine and thymine to protect cells from the potent effects of alkylating agents [101,103]. Hence, SNAP-tag performs a nucleophilic substitution reaction resulting in an irreversible, covalent coupling of BG-modified substrates, such as a fluorochrome, PS, or small molecule toxin with the thiol group of Cysteine 145, within the active site of the SNAP-tag molecule [103,104] (Figure 2).…”