Skip to main content
. 2017 Feb 21;56(11):1585–1595. doi: 10.1021/acs.biochem.7b00056

Figure 5.

Figure 5

No-wash imaging of Halo-tagged proteins in live cells using P9. (a) No-wash imaging of Halo-tagged cytosolic superoxide dismutase (SOD1) in HEK293T cells after labeling with P9. SOD1-Halo in the cytoplasm of transfected cells can be selectively imaged by P9 (2.5 μM) without a wash step (left lane). The Halo-tag TMR ligand (2.5 μM) exhibits a high fluorescence background without inclusion of washing steps (middle lane). The background can be eliminated by rinsing the cell with fresh medium (right lane). (b) No-wash imaging of Halo-tagged nuclear TAR DNA binding protein 43 (TDP43) in HEK293T cells using P9. TDP43-Halo is visible in the nuclei of transfected cells prior to washing using P9, whereas TDP43-Halo cannot be visualized clearly under the same conditions using TMR ligand (middle lane). HEK293T cells were transiently transfected with SOD1-Halo or TDP43-Halo for 24 h in 35 mm poly-d-lysine-coated glass bottom dishes. P9 and the Halo-tag TMR ligand (2.5 μM) were directly dissolved in the medium. Confocal images were taken after incubation for 30 min at 37 °C. The TMR-ligand-treated samples were washed further and incubated in fresh DMEM for an additional 30 min at 37 °C prior to confocal imaging. Hoechst 33342 is a nuclear staining dye.