Labeling of Proteins in vitro (S9220)

Protocol

1. Dissolve the vial of CLIP-Cell Block (100 nmol) in 50 μl of fresh DMSO to yield a labeling stock solution of 2 mM CLIP-Cell Block. Mix by vortexing for 10 minutes until all the CLIP-tag substrate is dissolved. Dilute this 2 mM stock solution 1:4 in fresh DMSO to yield a 500 μM stock for labeling proteins in vitro.
2. Set up the reactions, in order, as follows:

   Component	Volume	Final Concentration
   Deionized Water	30 μl
   5X CLIP-tag Reaction Buffer	10 μl	1X
   50 mM DTT	1 μl	1 mM
   50 µM CLIP-tag Purified Protein	5 μl	5 µM
   500 µM CLIP-Cell Block	2 μl	20 µM
   250 µM CLIP-tag Substrate	2 μl	10 µM
   Total Volume	50 μl

3. Incubate sample containing only 20 μM CLIP-Cell Block in the dark for 20 minutes at 37°C.
4. Once incubation with CLIP-Cell Block is complete, add 2 μl of 250 μM CLIP-tag substrate, mix and incubate in the dark for 60 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 the unreacted substrate can be seperated from the labeling CLIP-tag fusion protein by gel filtration or dialysis. Please refer to the vendor's instructions for the separation tools you are using.
   
   Notes 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, if handling at temperatures above 4°C is minimized.
   CLIP-tag fusion proteins can be purified before labeling, but the labeling reaction also works in non-purified protein solutions (including cell lysates).
   
   Troubleshooting
   For troubleshooting please refer to the instructions supplied with CLIP-Cell products as appropriate.