Figure 2.

Example of expected outcome for the SWFTI assay

A TIM tagged with an HA tag and SNAP tag was used as the bait to pull down a myc- and CLIP-tagged CRY in a bait-and-prey style immunoprecipitation. Both CRY and TIM were incubated together with HA resin with TIM as the bait. SNAP-Cell 647-SiR dye was utilized for SNAP-tagged TIM detection. The SNAP tag is excited at 647 nm and fluorescence is detected at a gel position corresponding near 120 kD, matching the expected molecular weight for the SNAP-tagged TIM protein being probed (pink). The CLIP-tagged CRY was visualized by exciting the CLIP-Cell 505 dye at 495 nm. Detection at expected excitation wavelength indicates positive protein detection from a stain-free gel. The data indicates that more CRY is bound to TIM in the light (L) compared to in the dark or absence of irradiation (D). In the assay shown, PBS was used to wash the resin instead of the suggested TBST resulting in less TIM visualized in the pull-down relative to the lysate due to protein aggregation on the resin.