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 (excitation/emission max)

Stock Concentration

How to Make

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

  1. After 30 minutes, remove the Snap-working solution and wash 3 times with FBS containing medium.

  2. 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.

  3. 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