===================================== Snap-tag Labeling ===================================== Cell expressing the Snap protein or a Snap-tagged fusion protein can be labeled live, without fixation using Snap ligands ordered from NEB. This protocol for labeling of live cells with Snap ligand is also found on `the NEB website `_. Snap-tag cell permeable dyes available in lab ________________________________________________ ======================= ========================== ========================= ========================================================================================================================================== **Dye** **Attune Channel** **Stock Concentration** **How to Make** (excitation/emission max) ======================= ========================== ========================= ========================================================================================================================================== Snap-Cell 430 VL1 (421/444) 1 mM (200x) Dissolve 1 vial of Snap-ligand in 50 µL of DMSO. Mix thoroughly by pipetting. Snap-Cell TMR-Star YL1 (554/580) 0.6 mM (200x) Dissolve 1 vial of Snap-ligand in 50 µL of DMSO. Mix thoroughly by pipetting. Snap-Cell 647-SIR RL1 (645/661) 0.6 mM (200x) Dissolve 1 vial of Snap-ligand in 50 µL of DMSO. Mix thoroughly by pipetting. ======================= ========================== ========================= ========================================================================================================================================== .. note:: Resuspended Snap-ligand is kept frozen in the -20°C freezer and has been used after several freeze/thaw cycles with no apparent detriment. .. note:: The NEB website recommends the above stock concentrations as 200x. Greater dilutions can been used and labeling is still sufficient. AMB has used 400x dilution (2.5 µL dye / mL media ) to label and had detectable signal. Greater dilutions may also reduce the amount of background staining. Other materials needed: ________________________ - DMEM + 10% FBS - Adherent cells to stain Add Snap ligand to cells -------------------------------------------- 1. Working in a BSC hood, dilute resuspended Snap ligand in an FBS containing medium 1:200 to making the Snap-working solution. Greater dilutions can be used for high-abundance proteins. 2. Aspirate media from adherent cells and replace media with the Snap-working solution. - For the Snap staining a "half" well volume may be used (e.g. 1 mL per well of 6-well plate, 0.5 mL in a 12-well, etc.) 3. Incubate the cells in Snap-working solution in the incubator (37°C) for 30 minutes. Wash and destain ligand -------------------------- 4. After 30 minutes, remove the Snap-working solution and wash 3 times with FBS containing medium. 5. On the third wash, leave the media on the cells and incubate at 37°C for 30 minutes to allow unreacted ligand to diffuse out. 6. Remove final wash media and proceed to downstream application (imaging, flow, etc.) .. note:: If you got high background signal, you can dilute resuspended Snap ligand in an FBS containing medium 1:500-1:1000