Detection by Fluorescent Imager (P9311)
- Add an appropriate volume of 3X 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 the appropriate excitation wavelength for the CLIP-tag fluorescent substrate used (e.g. 488 nm with CLIP-Vista Green).
- After fluorescent imaging, standard fixing and staining protocols can be used to detect any non-fluorescent proteins.
Most gel fixing/staining protocols will affect the fluorescence of the CLIP-tag substrates. The fluorescent gel should be appropriately scanned 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 Purified Protein. This will enhance the labeling by improving the stability and reactivity of the CLIP-tag protein. Labeling also works under non-reducing conditions. Care should be taken to avoid handling the CLIP-tag Purified Protein above 4°C prior to labeling.
Correct storage and handling of unlabeled CLIP-tag Purified Protein or CLIP-tag fusion proteins is essential to maintain the reactivity of the CLIP-tag prior to labeling. Unlabeled CLIP-tag Purified Protein or CLIP-tag fusion proteins should be stored at -20°C or -80°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-tag fluorescent substrate (e.g. CLIP-Vista Green) will run in front of the protein bands in the gel, running at an equivalent molecular weight below 20 kDa (below the band obtained for CLIP-tag alone). If the fluorescence from the unreacted substrate interferes with the 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 7.0–8.0). The salt concentration (50 mM–250 mM) and use of non-ionic detergent may also need to be optimized for your particular fusion protein.
If stickiness of the CLIP-tag Purified Protein or CLIP-tag fusion protein is a problem, we recommend adding Tween 20 to a final concentration of 0.05% to 0.1%. The CLIP-tag activity is not affected by this concentration of Tween 20.