Preparation of Labeling Stock Solution
Dissolve one vial of CLIP-tag substrate in 10 µl of fresh DMSO to yield a labeling stock solution of 1 mM CLIP-Cell 505 or 0.6 mM CLIP-Cell TMR-Star. Mix by vortexing for 10 minutes until all the CLIP-tag substrate is dissolved. Store this stock solution in the dark at 4°C, or for extended storage at –20°C. Different stock concentrations can be made, depending on your requirements. The substrates are soluble up to at least 10 mM.
Protocol for Intracellular Labeling Reaction
1. Dilute the labeling stock solution 1:200 in medium to yield a labeling medium of 5 µM CLIP-Cell-505 or 3 µM CLIP-Cell TMR-Star. Mix dye with medium thoroughly by pipetting up and down 10 times (necessary for reducing backgrounds). For best performance, add the CLIP-tag substrate to complete medium, including serum (0.5% BSA can be used for experiments carried out in serum-free media). Do not prepare more medium with CLIP-tag substrate than you will consume within one hour.
|NUMBER OF WELLS IN PLATE
||RECOMMENDED VOLUME FOR CELL LABELING
These recommendations are for culturing cells in polystyrene plates. For confocal imaging, we recommend using chambered coverglass such as Lab-Tek II Chambered Coverglass which is available in a 1, 2, 4 or 8 well format from Nunc (www.nuncbrand.com
2. Replace the medium on the cells expressing a CLIPf fusion protein with the CLIP-tag labeling medium and incubate at 37°C, 5% CO2 for 30 minutes.
3. Wash the cells three times with tissue culture medium containing serum and incubate in fresh medium for 30 minutes. Replace the medium one more time to remove unreacted CLIP-tag substrate that has diffused out of the cells.
4. Image the cells using an appropriate filter set. CLIPf
fusion proteins labeled with CLIP-Cell 505 should have an excitation maximum at 504 nm and an emission maximum at 532 nm, and can be imaged with standard fluorescein filter sets. CLIPf
fusion proteins labeled with CLIP-Cell TMR-Star should have an excitation maximum at 554 nm and an emission maximum at 580 nm, and can be imaged with standard rhodamine filter sets.