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. 2022 Jan 25;11:e74326. doi: 10.7554/eLife.74326

Figure 2. Using NbVHH05 and Nb127D01 for immunofluorescence.

(A) Fluorophore-conjugated NbVHH05 or Nb127D01 recognizes VHH05- or 127D01-tagged fluorescence proteins. (A’) VHH05- or 127D01-tagged mito-GFP can be detected by the corresponding NbVHH05-555 or Nb127D01-647 in transfected S2R+ cells. 4′,6-Diamidino-2-phenylindole (DAPI) staining shows the nuclei. (B) Schematic of nanobodies containing ALFA-tag as primary antibody and NbALFA as a secondary antibody. (B’) VHH05- or 127D01-tagged mito-GFP can be detected using the corresponding nanobodies in transfected S2R+ cells. (C) Schematic of fluorophore-conjugated anti-Alpaca IgG antibodies to detect VHH05- and 127D01-tagged proteins. NbVHH05 or Nb127D01 is used as primary antibodies and anti-Alpaca IgG as secondary antibody. (C’) VHH05- or 127D01-tagged mito-GFP can be detected using the corresponding nanobodies and anti-Alpaca IgG-647 in transfected S2R+ cells. (D) Schematic of using VHH05 and 127D01 for double tagging. N-, C-terminal of REPTOR contains VHH05 and 127D01. (D’) Co-staining NbVHH05 and Nb127D01 in S2R+ cells transfected with VHH05-REPTOR-127D01. Scale bars: 10 µm.

Figure 2.

Figure 2—figure supplement 1. Nanobody purification and fluorophore conjugation.

Figure 2—figure supplement 1.

(A) Fluorescence signals of NbVHH05-CF555 and Nb127D01-CF647 under 555 and 647 detection channels. (B and C) Coomassie brilliant blue staining of ALFA- and HA-tagged nanobodies purified from Escherichia coli protein expression. FT: flow-through, E1–E9: elution 1–9.
Figure 2—figure supplement 1—source data 1. Raw data of fluorescence signals or coomassie brilliant blue staining for Figure 2—figure supplement 1.
Figure 2—figure supplement 2. Different types of NbVHH05 and Nb127D01 and immunofluorescence examples.

Figure 2—figure supplement 2.

(A) Direct labeling can be achieved by linking purified nanobodies with different fluorophores by antibody labeling kit or site-specifically by sortase-mediated labeling. (A1) Schematics of NbVHH05 and Nb127D01 with fluorophore-488, -555, or -647. (A2) Confocal images showing direct immunofluorescence with NbVHH05-555, NbVHH05-488, and Nb127D01-647 in S2R+ cells. (A3) Confocal images showing direct immunofluorescence with NbVHH05-555 prepared by sortase-mediated labeling. (B) Schematics of indirect labeling with nanobodies containing ALFA-tag, HA-tag, biotin, or human IgG as primary antibodies and confocal images in S2R+ cells. (B1) NbVHH05 and Nb127D01 detected with anti-VHH IgG antibody. (B2) NbVHH05 and Nb127D01 with ALFA-tag detected by NbALFA. (B3) NbVHH05 and Nb127D01 with HA-tag detected by anti-HA antibody. (B4) NbVHH05 and Nb127D01 with biotin prepared by sortase-mediated labeling detected by streptavidin. (B5) NbVHH05 and Nb127D01 with human IgG detected by anti-human IgG antibody. Scale bars: 10 µm.
Figure 2—figure supplement 3. Test of potential interaction between VHH05 and 127D01.

Figure 2—figure supplement 3.

(A) Fluorescence confocal results showed no co-localization signal. Co-transfection of Nb127D01-GFP and mito-mCherry-VHH05 or H2B-mCherry-VHH05, NbVHH05-GFP and mito-mCherry-127D01 or H2B-mCherry-127D01 in S2R+ cells. (B) Western blots indicate no cross-interaction between the two systems. Lysates from S2R+ cells transfected with different types tagged vectors (as in Figure 1) or a mock control plasmid were analyzed by SDS-PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody, and followed by anti-mouse IgG HRP.
Figure 2—figure supplement 3—source data 1. Raw data of Western blot for Figure 2—figure supplement 3b.