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  • Labeling of Proteins in vitro (E9100)

    Introduction

    The supplied dye substrates can also be used to label SNAP-tag fusion proteins in solution for biochemical applications including FRET, labeling of proteins used for detection, etc.

    1. Dissolve the vial of SNAP-Cell 505 (10 nmol) in 10 µl of fresh DMSO or the vial of SNAP-Cell TMR-Star (6 nmol) in 6 µl of fresh DMSO to yield a labeling stock solution of 1 mM SNAP-tag substrate. Mix by vortexing for 10 minutes until all the SNAP-tag substrate is dissolved. Dilute this 1 mM stock solution 1:4 in fresh DMSO to yield a 250 µM stock for labeling proteins in vitro.

    2. Set up the reactions, in order, as follows:

    3. COMPONENT VOLUME FINAL CONCENTRATION
      Phosphate Buffered Saline (PBS) 42 µl 1X
      50 mM DTT 1 µl 1 mM
      50 µM SNAP-tag Purified Protein 5 µl 5 µM
      250 µM SNAP-tag Substrate 2 µl 10 µM
      Total Volume  50 µl

    4. Incubate in the dark for 15 minutes at 37°C.

    5. 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.

    Removal of Unreacted Substrate (optional)

    After the labeling reaction you may wish to separate the nonreacted substrate from the labeled SNAPf fusion protein. You can use gel filtration or dialysis. Please refer to the vendor’s instructions for the separation tools you are using.

    Note for Labeling in vitro

    We recommend the routine addition of 1 mM DTT to all buffers used for handling, labeling, and storage of the SNAP-tag. The stability of the SNAP-tag is improved in the presence of reducing agents; however it can also be labeled in their absence (e.g. for a redox-sensitive protein) if handling at temperatures above 4°C is minimized. SNAPf  fusion proteins can be purified before labeling, but the labeling reaction also works in non-purified protein solutions (including cell lysates).