Overall, the click chemistry-based method may be an attractive alternative method for the detection of TG2 activity and/or substrates because of the advantage of the relatively small size of the amine-donor substrate and the introduction of a flexible labeling step after the crosslinking of the substrate, allowing differential labeling of cellular proteins.
We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators.
As expected, α-α homodimers were not detected in a cell line expressing only the mammalian α-subunit [35S] labeling of cellular proteins (Fig. S5A).
If the avian (chicken) α-subunit alone was expressed, low, but detectable, αChwt/αChwt mature homodimers were detected both by immunoblot and autoradiography following [35S] labeling of cellular proteins (Fig. 3B and S5A), and were confirmed by MS-MS (Figs. S5B and S5C).
Site-specific labeling of cellular proteins with chemical probes is a powerful tool for live cell imaging of biological processes.
This new method for labeling TG2 substrate proteins introduces flexibility in the detection and/or purification of crosslinked proteins, allowing differential labeling of cellular proteins.
The plug-ins made it possible to evaluate clustering and co-localization patterns detected by immunogold labeling, and to identify and map significant labeling of cellular compartments.
The carborhodamine label displayed similar localizations (842,372/cell) to the genetically encoded mEos2 (1,011,424/cell), showing comparable labeling of cellular actin with these two fluorophores.
Although this method provides efficient measurement of efflux when compared with the use of radiolabeled cholesterol, it still requires labeling of cellular sterols [139].
For labeling of cellular proteins, HeLa cells were incubated in medium without methionine and supplied with 2% dialyzed fetal calf serum (FCS).
