In many cases the labeling of a non-transfected cell sample or a mock-transfected cell sample will be completely sufficient as a negative control for cell labeling. In some cases, however, it may be desirable to block the SNAP-tag activity in a cell sample expressing the SNAPf fusion protein to generate a control. This is done by a pre-incubation of the cells with SNAP-Cell Block, followed by the incubation with the labeling solution. SNAP-Cell Block may also be used in pulse-chase experiments to block the SNAP-tag reactivity during the chase between two pulse-labeling steps.
Note that SNAP-Cell Block is a potent blocker of the SNAP-tag. Always take care to avoid carryover of SNAP-Cell Block to samples that you do not wish to block.
Preparation of Stock Solution
Dissolve one tube of SNAP-Cell Block (20 nmol) in 10 μl of fresh DMSO to give a stock solution of 2 mM. Mix by vortexing for 10 minutes, until all the SNAP-Cell Block is dissolved. Store this stock solution in the dark at 4°C or for extended storage at -20°C. We recommend using a final concentration of 10 μM, which is a 1:200 dilution of this stock solution.
Blocking SNAP-tag Activity with SNAP-Cell Block
The following steps describe the use of SNAP-Cell Block in a typical control labeling experiment:
- Prepare two cell samples suitable for labeling, each expressing the SNAPf fusion protein of interest.
- Mix an appropriate amount of medium with SNAP-Cell Block stock solution in a ratio of 1:200 to give a blocking medium of 10 μM SNAPCell Block. For best performance, add the dissolved SNAP-Cell Block to complete medium, including serum. Do not prepare more medium with SNAP-Cell Block than you will consume within one hour.
- Mix an appropriate amount of medium with DMSO in a ratio of 1:200, to give a final concentration of 0.5% v/v DMSO.
- Replace the medium on one sample of cells with the blocking medium. These are your blocked cells. Replace the medium on the other sample of cells with the medium containing DMSO. These are your test cells. Incubate both cell samples at 37°C, 5% CO2 for 10–15 minutes.
- Remove SNAP-Cell Block or DMSO-containing medium by washing both samples of cells twice with complete medium.
- Label both cell samples with the SNAP-Cell substrate using the Protocol for Intracellular Labeling Reaction.
- Inspect both samples under the fluorescence microscope. The blocked cells should show no fluorescence, whereas the test cells should show fluorescence localized to where the SNAPf fusion protein is present in the cell.
Note that there is a constant turnover and resynthesis of proteins in the cell. After having blocked all existing SNAPf fusion proteins within the cell, new SNAPf fusion protein molecules may be synthesized in the meantime and may get labeled during incubation with a fluorescent SNAP-tag substrate. This will give the impression that the blocking was ineffective. In order to minimize these effects of protein synthesis and protein transport, cells may have to be treated with cycloheximide and incubation with the fluorescent SNAP-tag substrate may have to be performed at 4°C.