Abstract
Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the “RAP tag” system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification.
Highlights
Affinity tag systems, which are useful for protein purification and detection, are classified into ‘‘peptide tags’’ and ‘‘protein tags.’’ Protein tags, including glutathioneS-transferase tag,(1) maltose-binding protein tag,(2) Fc tag of immunoglobulin,(3) and green fluorescent protein tag,(4) have numerous advantages because they are useful for protein expression in the soluble fraction or are detected using monoclonal antibodies
We previously developed a mouse monoclonal antibodies (mAbs) against the platelet aggregation-stimulating domain of rat podoplanin.[24]. Podoplanin is a type I transmembrane protein, which is highly expressed in many normal cells and cancer cells, and is involved in tumor-induced platelet aggregation by binding to CLEC-2 on platelets.[3,25,26,27,28,29,30,31,32,33] Because PMab-2 possesses high affinity and specificity against rat podoplanin,(24) it was expected to be useful as an antitag antibody
Curve fitting showed the affinity for PMab-2– RAP tag interaction: ka = 2.0 · 105 M-1 s-1, kd = 2.0 · 10-3 s-1, and KD = 9.7 · 10-9 M, indicating that PMab-2 showed moderate affinity toward the RAP tag
Summary
Affinity tag systems, which are useful for protein purification and detection, are classified into ‘‘peptide tags’’ and ‘‘protein tags.’’ Protein tags, including glutathioneS-transferase tag,(1) maltose-binding protein tag,(2) Fc tag of immunoglobulin,(3) and green fluorescent protein tag,(4) have numerous advantages because they are useful for protein expression in the soluble fraction or are detected using monoclonal antibodies (mAbs). Peptide tags are less likely to affect the structure and function of target proteins because of their small size (typically 1–2 kDa); it is not always necessary to remove the tag portion for protein analysis. We previously developed a mouse mAb (clone PMab-2) against the platelet aggregation-stimulating domain of rat podoplanin.[24] Podoplanin is a type I transmembrane protein, which is highly expressed in many normal cells and cancer cells, and is involved in tumor-induced platelet aggregation by binding to CLEC-2 on platelets.[3,25,26,27,28,29,30,31,32,33] Because PMab-2 possesses high affinity and specificity against rat podoplanin,(24) it was expected to be useful as an antitag antibody.
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