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 selectively block the SNAP-tag activity on the cell-surface 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-Surface Block, followed by the incubation with the labeling solution. Always take care to avoid carryover of SNAP-Surface Block to SNAP-Surface 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-Surface Block should block >90% of active SNAP-tag on the cell surface under the conditions given below, however complete blocking is difficult to achieve. SNAP-Surface Block is slightly cell permeable so use of it may slightly reduce signal from intracellular tagged proteins in later labeling steps. This effect is increased when SNAP-Surface Block is used at higher concentrations and for longer incubation times. Always take care to avoid carryover of SNAP-Surface Block to samples that you do not wish to block.
The following steps describe the use of SNAP-Surface Block in a typical labeling experiment:
- Dissolve one vial of SNAP-Surface Block (200 nmol) by adding 50 µl of DMSO to give a solution of 4 mM SNAP-Surface Block. Mix by vortexing for 10 minutes, until all the SNAP-Surface 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 20 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 20 μM SNAP-Surface Block. Mix blocker with medium throughly by pipetting up and down 10 times. For best performance, add the dissolved SNAP-Surface 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-Surface 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 throughly 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.
|Number of wells in plate
||Recommended Volume for Cell Block
- These recommendations are for culturing cells in polystyrene plates. For confocal imaging, we recommend using chambered coverglass.
- Remove SNAP-Surface Block or DMSO containing medium by washing both samples of cells twice with complete medium.
- Label both cell samples with the SNAP-surface 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 on the cell surface where the SNAP-tag fusion protein is present.
Note: Please note that there is a constant turnover of proteins in the cell. Protein transport to the membrane and internalization followed by degradation or recycling, are constantly ongoing processes. After having blocked SNAP-tag on the cell membrane, newly synthesized protein may be transported to the cell surface 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.