Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes
Fig 1
Schematic illustration of the functional characteristics of annexin V-SNAP (AV-SNAP) and SDS-PAGE of AV-SNAP coupled to various BG-modified dyes.
(A) Simple and rapid labeling reaction of AV-SNAP to different BG-modified fluorescent dyes. The SNAP-tag technology demonstrates irreversible coupling of BG-modified dyes to AV-SNAP as a fusion protein. The labeling takes place in a 1:1 stoichiometry without inhibiting the fusion partner AV. During the labeling reaction, free guanine is released. (B) The principles of an AV apoptosis assay are demonstrated. On healthy cells with a normal cell membrane, phosphatidylserine (PS) is located on the inner surface. Phospholipids of the cell membrane are asymmetrically distributed between outer and inner cell membrane. During early stages of apoptosis, PS becomes exposed on the outer surface of the plasma cell membrane due to loss of plasma membrane asymmetry. PS exposed to the external cellular environment can be bound by AV which show high respective affinities and consequently the AV-SNAP-BG-dye coupled fusion protein can be used to detect apoptotic changes in target cells. In later stages of cell death, loss of membrane integrity occurs also, demonstrating late apoptotic as well as necrotic stages in cells. Typically, PI is used together with AV in the same assay, because PI is a vital dye and permeable cell membranes of damaged or dead cells are open for PI. (C) SDS-PAGE analysis of BG-fluorophore-labeled AV-SNAP. Coomassie Brilliant Blue staining (left) of the SDS PAGE and visualization of labeled fluorophores using the CRi Maestro imaging system (right) with the blue (500–720 nm) and yellow (630–850 nm) filter set. Dye spectra were unmixed using the associated software 2.2. (M) Color pre-stained protein standard; AV-SNAP labeled with (1) BG- 488, (2) BG-505, (3) BG-532, (4) BG-546 and (5) BG-647.