Labeling SNAP-tag Purified Protein In Vitro (P9312)
- Thaw the SNAP-tag Purified Protein or SNAP-tag fusion protein at room temperature just before use.
- Set up the reactions, in order, as follows:
Component Volume Final Concentration 1X PBS 42µl 1 X 50 mM DTT 1 µl 1 mM 50 µM SNAP-tag Purified Protein 5µl 5 µM 250 µM SNAP-tag substrate 2 µl 10 µM Total Volume 50µl
- Incubate in the dark for 30 minutes at 37°C.
- Run sample on an SDS-PAGE gel and detect using a fluorescent gel scanner or store samples at -20°C or -80°C in the dark.
Notes for In Vitro Labeling:
Adjust the volumes to achieve the final concentrations indicated if the stock solutions of protein, substrate, and DTT are different than described above.
One may choose to label the SNAP-tag Purified Protein or SNAP-tag fusion protein in a different final concentration, however, it is recommended to use a 2-fold excess of SNAP-tag substrate vs. SNAP-tag protein.
Labeling may also be carried out in 1X PBS solution or 50 mM HEPES containing 1 mM DTT.
If solubility problems occur, we recommend testing a range of pH (pH 7.0–8.0) and ionic strengths. 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 SNAP-tag Purified Protein or SNAP-tag fusion protein is a problem, we recommend adding Tween 20 to a final concentration of 0.05% to 0.1%. The SNAP-tag activity is not affected by this concentration of Tween 20.