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. 2020 Sep 12;21(18):6672. doi: 10.3390/ijms21186672

Figure 2.

Figure 2

Characterization of 5D3 antibody fragments expressed in E. coli. (A) Purity of recombinant proteins was estimated from SDS-PAGE gel stained by Coomassie Brilliant Blue G-250 (CBB). (B) Binding of 5D3 variants on live cells was estimated by flow cytometry using anti-6× His antibody together with an anti-mouse IgG-Alexa Fluor 647 conjugate. All molecules used at saturating concentrations showed specific staining of PSMA-positive LNCaP cells with similar signal intensity. PSMA-negative DU145 cells were used as a control. (C) Indirect immunofluorescence microscopy using 5D3 scFv LH. PSMA-overexpressing HEK293T cells and the parent HEK293T cell line (a negative control) were fixed with/without permeabilization by Triton X-100 and incubated with 5D3 scFv LH. The fragment was then detected by anti-6× His Ab combined with anti-mouse Alexa Fluor 488 conjugate (green channel). Cell nuclei were counterstained by DAPI (blue channel). The scale bar represents 25 µm.