Labeling Proteins in vitro (E9120)
- Dissolve one vial of SNAP-Surface 488 or SNAP-Surface 549 (10 nmol) in 10 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.
- Set up the reactions, in order, as follows:
COMPONENT VOLUME FINAL CONCENTRATION Deionized Water 32 µl 5X SNAP-tag Reaction Buffer 10 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
- Incubate in the dark for 15 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.
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).