Use with SNAP-Surface Substrates (E9120)
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 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. 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.
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.
Note that SNAP-Surface Block is a potent blocker of the SNAP-tag! Always take care to avoid carryover of SNAP-Surface Block to samples that you do not wish to block.
Preparation of Stock Solution
Dissolve one tube of SNAP-Surface Block (40 nmol) in 10 μl of DMSO to give a solution of 4 mM. Mix 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.
Blocking SNAP-tag Activity with SNAP-Surface Block
The following steps describe the use of SNAP-Surface Block in a typical control labeling experiment:
- Prepare two cell samples suitable for labeling, each expressing a surfacelocalized SNAP-tag fusion protein of interest.
- Mix an appropriate amount of medium with SNAP-Surface Block stock solution in a ratio of 1:200 to give a blocking medium of 20 μM SNAPSurface Block. For best performance, add the dissolved SNAP-Surface Block to complete medium, including serum. 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.
- 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.
- Remove SNAP-Surface Block or DMSO-containing medium by washing both samples of cells twice with complete medium.
- Label both cell samples with a SNAP-Surface substrate using the Protocol for Cell Surface 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 the cell surface where the SNAP-tag fusion protein is present.
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.
Cloning of the Gene of Interest
If subcloning of the gene of interest into the SNAP-tag vector does not work, reconfirm all the cloning steps (primer design, choice of restriction site, etc.). If all steps are confirmed as being correct, then try the cloning using different restriction sites. Be sure to include a positive control for the ligation reaction. Alternatively try to subclone the SNAP-tag gene into an expression vector already containing your gene of interest.
In general we have not experienced problems expressing SNAP-tag protein fusions. However if your fusion protein does not appear to be expressed, try expressing the SNAP-beta-2 adrenergic receptor protein fusion as a positive control using cells transiently transfected with the included pSNAP-ADRB2 control plasmid. Labeling of such cells with a fluorescent SNAP-Surface substrate should show strong surface-localized fluorescence. The empty pSNAPm plasmid can also be used as a control (uniform cytosolic and nuclear fluorescence when using cell-permeable SNAP-Cell substrates). Note that the intensity of this fluorescence may vary depending on cell line and substrate used. Expression of localization controls but not your fusion protein can be due to a variety of causes. It is possible that this fusion protein may be toxic for your cell line. It is difficult to troubleshoot such instances, but the use of a different expression plasmid or cell line or tagging the opposite end of the protein may help. Signs of host cell toxicity could include slow proliferation or apoptosis. Counterstaining live cells with Hoechst 33342 or fixed cells with DAPI can be used to determine whether nuclei are healthy if toxicity is suspected.