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 CLIP-tag activity in a cell sample expressing the CLIPf fusion protein to generate a control. This is done by a pre-incubation of the cells with CLIP-Cell Block, followed by the incubation with the labeling solution. CLIP-Cell Block may also be used in pulse-chase experiments to block the CLIP-tag reactivity during the chase between two pulse-labeling steps.
Note that CLIP-Cell Block is a potent blocker of the CLIP-tag! Always take care to avoid carryover of CLIP-Cell Block to samples that you do not wish to block.
Preparation of Stock Solution
Dissolve one tube of CLIP-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 CLIP-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 CLIP-tag Activity with CLIP-Cell Block
The following steps describe the use of CLIP-Cell Block in a typical control labeling experiment:
- Prepare two cell samples suitable for labeling, each expressing the CLIPf fusion protein of interest.
- Mix an appropriate amount of medium with CLIP-Cell Block stock solution in a ratio of 1:200 to give a blocking medium of 10 μM CLIP-Cell Block. For best performance, add the dissolved CLIP-Cell Block to complete medium, including serum. Do not prepare more medium with CLIP-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 30 minutes.
- Remove CLIP-Cell Block or DMSO-containing medium by washing both samples of cells twice with complete medium.
- Label both cell samples with the CLIP-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 where the CLIPf fusion protein is present in the cell.
Note that there is a constant turnover and resynthesis of proteins in the cell. Protein transport to the membrane and internalization followed by degradation or recycling, are constantly ongoing processes. After having blocked all existing CLIPf fusion proteins on the cell membrane, newly synthesized protein may be transported to the cell surface and may get labeled during incubation with a fluorescent CLIP-Surface 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 CLIP-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 pCLIPf 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 and negative control for the ligation reaction. Alternatively try to subclone the CLIPf gene into an expression vector already containing your gene of interest.
In general we have not experienced problems expressing CLIPf protein fusions. However if your fusion protein does not appear to be expressed, try expressing the CLIPf-NK1R protein fusion as a positive control using cells transiently transfected with the included pCLIPf-NK1R. Labeling of such cells with a fluorescent CLIP-Surface substrate should show strong surface-localized fluorescence. The empty pCLIPf plasmid can also be used as a control (uniform cytosolic and nuclear fluorescence when using cell-permeable CLIP-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.