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 SNAP-tag 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: 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.
The following steps describe the use of SNAP-Cell Block in a typical control labeling experiment:
- Dissolve one vial of SNAP-Cell Block (100 nmol) by adding 50 µl of DMSO to give a solution of 2 mM SNAP-Cell Block. 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.
- Prepare two cell samples suitable for labeling, expressing the SNAP-tag fusion protein of interest.
- Dilute the blocking stock solution 1:200 in medium to yield a blocking medium of 10 µM SNAP-Cell Block. Mix blocker with medium thoroughly by pipetting up and down 10 times. For best performance, add the dissolved SNAP-Cell Block to complete medium, including serum (0.5% BSA can used for experiments carried out in serum-free media). 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. Mix thoroughly by pipetting up and down 10 times.
- 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 for 20 minutes.
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.
|Number of wells in plate
||Recommended Volume for Cell Labeling
- Remove SNAP-Cell Block or DMSO-containing medium by washing both samples of cells twice with complete medium.
- Label both cell samples with the fluorescent SNAP-Cell substrate using the supplied protocol.
- 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 SNAP-tag fusion protein is present in the cell.
Note: Please note that there is a constant turnover and resynthesis of proteins in the cell. After having blocked all existing SNAP-tag fusion proteins within the cell, new SNAP-tag 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.