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 CLIP-tag activity in a cell sample expressing the CLIP-tag 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: under the conditions given below, CLIP-Cell Block blocks most of the active CLIP-tag. However, achievement of complete blocking may be dependent on experimental conditions. Always take care to avoid carryover of CLIP-Cell Block to samples that you do not wish to block.
The following steps describe the use of CLIP-Cell Block in a typical labeling experiment:
- Dissolve one tube of CLIP-Cell Block (100 nmol) by adding 50 µl of DMSO to give a solution of 2 mM CLIP-Cell Block in DMSO. 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.
- Prepare two cell samples suitable for labeling, expressing the CLIP-tag 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 for 30 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 Block
- Remove CLIP-Cell Block or DMSO containing medium by washing both samples of cells twice with complete medium.
- Label both cell samples with the fluorescent CLIP-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 CLIP-tag fusion protein is present in the cell.