Labeling Purified Proteins in vitro (S9235)
- Add 2 µl of the substrate stock solution to 18 µl of protein sample containing a CLIP-tag fusion protein in an appropriate buffer (see notes). Mix well by pipetting up and down several times.
- Incubate in the dark for 60 minutes at 37°C.
- Add an appropriate volume of concentrated SDS-PAGE sample buffer and proceed with sample preparation and SDS-PAGE according to the gel manufacturer’s instructions.
- After the gel is run, immediately take a fluorescent image using a laser scanner with 488 nm excitation or a UV-transilluminator and an appropriate camera (Polaroid or digital). Excitation at 488 nm will give the best results. The fluorescence is an intense green.
- After fluorescent imaging, standard fixing and staining protocols can be used to detect the non-fluorescent proteins.
CLIP-Vista Green is dried down with mannitol to improve its aqueous solubility. Mannitol will be present at 10 mM final concentration and should not lead to any problems.
Most gel fixing/staining protocols will affect the fluorescence of the CLIP-Vista substrate. The fluorescent gel image should be appropriately documented before continuing with protein staining.
We recommend the routine addition of 1 mM DTT to all buffers used for handling, labeling and storage of the CLIP-tag. This will enhance the labeling by improving the stability and reactivity of the CLIP-tag fusion protein. Labeling also works under non-reducing conditions. Care should be taken to avoid handling the CLIP-tag fusion protein above 4°C prior to labeling.
Where stickiness of the fusion protein is a problem we recommend adding Tween 20 at a final concentration of 0.05% to 0.1%. At this concentration Tween 20 does not affect the performance of the CLIP-tag.
Correct storage and handling of unlabeled CLIP-tag fusion proteins is essential to maintain reactivity of the CLIP-tag prior to labeling. Unlabeled fusion proteins should be stored at -20°C and thawed just before use. Prolonged handling at temperatures above 4°C should be avoided, especially if the protein is stored in the absence of reducing agents (e.g., DTT).
The CLIP-tag labeling reaction is tolerant of a wide range of buffers. The requirements of the fusion partner should dictate the buffer selected. The following buffer guidelines are recommended: pH between 7.0 and 8.0, monovalent salts (e.g. sodium chloride) between 50 mM and 250 mM, at least 1 mM DTT. Non-ionic detergents can be added to 0.5% v/v if required, but SDS and other ionic detergents should be avoided entirely because they inhibit the activity of the CLIP-tag. Metal chelating reagents (e.g., EDTA and EGTA) also inhibit CLIP-tag activity and should be avoided. Many proteins benefit from the addition of glycerol for frozen storage, typically 20% v/v.
Unreacted CLIP-Vista substrate will run in front of the protein bands in the gel, running at an equivalent molecular weight below 10 kDa (below the band obtained for CLIP-tag alone). If the fluorescence from the unreacted substrate interferes with imaging for your protein, you may separate the labeled protein from unreacted substrate after the labeling reaction and before running the gel using, for example, a spin separation device.
If solubility problems occur, we recommend testing a range of pH (pH 7.0–pH 8.0) and ionic strengths. The salt concentration (50–250 mM) may also need to be optimized for your particular fusion protein.
If stickiness of the fusion protein is a problem we recommend adding Tween 20 at a final concentration of 0.05% to 0.1%. The CLIP-tag activity is not affected by this concentration of Tween 20.