Abstract
The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research.
Highlights
During characterization of the Monoclonal antibodies (mAbs) by Western blotting, we noticed that mAb G196 recognized the proteins encoded by pGEX-4T-2 (4T-2, Fig. 1a) and by pGEX-2T, but not the proteins encoded by pGEX-6P-1 (6P-1, Fig. 1a) or by pGEX-3X
MAb G196 failed to detect the core domain of GST protein (2–212), indicating that the G196 epitope falls within the overlapping C-terminal extension of the proteins encoded by pGEX-2T and pGEX-4T-2 (Fig. 1a, lower panel)
MAb G196 recognized G196- and GFP-tagged Atf[1], as shown by immunofluorescent staining of yeast, comparable to that of a polyclonal anti-GFP antibody (Fig. 5d). These results indicate that the G196 epitope tag system is suitable for Western blotting, immunoprecipitation, chromatin immunoprecipitation (ChIP), and immunofluorescence assay in both yeast and human
Summary
We applied the G196 epitope tag system to immunofluorescence assays with HeLa cells expressing the G196-tagged reporter protein to evaluate its utility. MAb G196 successfully detected the nuclear reporter protein, as did the control polyclonal anti-GFP antibody (Fig. 5b).
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