Labeling SNAP-tag Purified Protein In Vitro (P9312)

Protocol

1. Thaw the SNAP-tag Purified Protein or SNAP-tag fusion protein at room temperature just before use.
   
   
2. 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

3. Incubate in the dark for 30 minutes at 37°C.
   
   
4. 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. 
   
   Troubleshooting 
   
   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.