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Fig. 2 | Biomaterials Research

Fig. 2

From: CDH17 nanobodies facilitate rapid imaging of gastric cancer and efficient delivery of immunotoxin

Fig. 2

Characterization of A1 and E8 nanobodies against CDH17. a SAD-PAGE analysis of purified nanobodies (left) and nanobody confirmation with HA antibody (middle) and His antibody (right). The molecular weight for three nanobodies ranged from 15 kDa to 18 kDa. b ELISA analysis of binding ability of A1 and E8 nanobodies to CDH17 domain 1-3 (n = 2). Data are representative of two independent experiments. c Determination of binding affinity to CDH17 protein by SPR analysis. The equilibrium dissociation constant KD was 377 nM (A1) and 70.3 nM (E8) respectively. d Binding activity of A1 and E8 nanobodies in CDH17-positve cells (MKN45, IM95, TMK1 and AGS) assessed by fluorescent cell ELISA (n = 3). Both of nanobodies could recognize CDH17 protein expressed in cell membrane, while E8 nanobody shows a better performance. Data are expressed as mean ± SEM. e Validation of knockdown CDH17 with shRNA#3 in IM95 and MKN45 cells determined with western blot. f Binding specificity of E8 nanobody to CDH17 in CDH17-overexpressing and -knockdown cell lines respectively. E8 nanobody cannot obviously stain the cells with knockdown CDH17, indicating the great specificity of E8 nanobody to CDH17. g Internalization analysis of E8 nanobody with one-hour and three-hour incubations followed with HA-tag staining. An irrelevant nanobody as a control was used for all the assays above

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