Home Protocols Labeling of Proteins in vitro (S9221)

Labeling of Proteins in vitro (S9221)

  1. Dissolve the vial of CLIP-Biotin (50 nmol) in 50 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.

  2. Set up the reactions, in order, as follows:
    1. Component
    		Volume Final
    Concentration
    Phosphate Buffered Saline (PBS) 42 μl 1X
    50 mM DTT 1 μl 1 mM
    50 µM CLIP-tag
    Purified Protein    		 5 μl 5 µM
    250 µM CLIP-tag
    Substrate    		 2 μl 10 µM
    Total Volume 50 μl  

  3. Incubate in the dark for 60 minutes at 37°C.

  4. Run sample on an SDS-PAGE gel and detect using standard streptavidin-based detection reagents 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 separated from the labeled 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 

    Labeling Reaction
    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).

    Confirmation of Labeling by Western blot Analysis
    Labeled CLIP-tag fusion proteins can be easily analyzed on a SDS-PAGE gel/Western blot analysis because the covalently bound label will remain attached to the protein. The biotin label can be detected on an SDS-PAGE gel followed by Western Blot using a Horseradish Peroxidase or Alkaline Phosphatase labeled avidin/streptavidin (e.g. streptavidin-HRP) and the corresponding detection method as described by the supplier of the enyzme conjugate.