Labeling Proteins in vitro (E9200)
- Dissolve the vial of CLIP-Cell 505 (10 nmol) in 10 µl of fresh DMSO or the vial of CLIP-Cell TMR-Star (6 nmol) in 6 µl of fresh DMSO to yield a labeling stock solution of 1 mM CLIP-tag substrate. Mix by vortexing for 10 minutes until all the CLIP-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 CLIP-tag Reaction Buffer 10 µl 1X 50 mM DTT 1 µl 1mM 50 µM CLIP-tag Purified Protein 5 µl 5 µM 250 µM CLIP-tag Substrate 2 µl 10 µM Total Volume 50 µl
- 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.
After the labeling reaction you may wish to separate the nonreacted substrate from the labeled CLIPf 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 CLIP-tag. The stability of the CLIP-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. CLIPf fusion proteins can be purified before labeling, but the labeling reaction also works in non-purified protein solutions (including cell lysates).