Labeling proteins inside living cells using external fluorophores for microscopy
- University of Illinois at Urbana-Champaign, United States
Figures
Figure 1 with 1 supplement
Fluorescent labeling of proteins inside the living cell with high efficiency.
(a) Schematic of permeabilization, delivery, for reversible permeabilization using streptolysin O (SLO) on live CHO-K1 cells. Cells are exposed to SLO for 7–10 min before incubating with the three …
Figure 1—figure supplement 1
Propidium iodide exclusion and cell division assay.
(a) Propidium iodide assay performed on CHO-K1 cell after SLO permeabilization and recovery. The CHO-K1 cells were recovered in different buffers after permeabilization. Only recovery media and 10% …
Figure 2 with 1 supplement
Labeling cellular proteins with a variety of probes.
(a) Delivery of different sized fluorescent probes for specific labeling of intracellular proteins. The drawings depicting the fluorescent probes are not to scale. (b) Cells are labeled by various …
Figure 2—figure supplement 1
The effect of fluorescent probe sizes on retention and nucleus permeability.
(a) Size cut-off for nucleus accessibility determined using different sized FITC-dextran. We tested the molecular weight cutoff for nucleus entry with fluorescein dextran on CHO-K1 cells. We …
Figure 3 with 3 supplements
Applications of live cell protein labeling using SLO permeabilization.
(a) The number of detected single molecule activation events per frame with and without the addition of glutathione in the recovery media. With the addition of 4 mM glutathione in the recovery media …
Figure 3—figure supplement 1
Activation of Alexa647 in fixed and live cells in the presence and absence of Oxyrase.
(a) Representative images of Alexa647 de-activation. U2OS cells were fixed and labeled with Alexa647 to confirm the condition for photoactivation. The cells were fixed in 4% paraformaldehyde and …
Figure 3—figure supplement 2
Additional examples of intracellular structures of living cells imaged by dSTORM using cell impermeant chloroalkane-dye that are delivered by using SLO.
On the left is a dSTORM image of Histone 2B protein in the nucleus of HeLa cells. The H2B was labeled by Alexa647-chloroalkane. The image on the right hand side is a dSTORM image of mitochondria …
Figure 3—figure supplement 3
Labeling fluorescent protein with conjugated nanobody for kinesin tracking.
(a) Improving the detection of proteins attached to fluorescent protein by using nanobody labeling. 6xHIS-GFP was immobilized on glass coverslip using the described schematic. ATTO647N-GBP was …
Videos
Video 1
Dynamic direct super-resolution imaging of actin labeled with Alexa647-phalloidin related to Figure 3.
Real time is displayed at the bottom right hand corner of the movie in min:s.
Video 2
Single molecule detection of individual mCherry kinesin using ATTO647N labeled anti-RFP nanobody.
Real time is displayed at the bottom right hand corner of the movie in min:sec, the movie is played back in 3x the speed compared to real time.
Tables
Table 1
Dilution and incubation time of SLO to use for cell lines used in this study at various confluency.
Cell line | Confluency | Dilution of SLO stock | Incubation time (min) |
|---|---|---|---|
CHO-K1 | 90–100% | 250x | 7 |
CHO-K1 | 75% | 334x | 7 |
U2OS | 80% | 500x | 7 |
U2OS | 50% | 774x | 7 |
U2OS | 30% | 1000x | 7 |
HeLa | 90% | 250x | 10 |
