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  • 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.