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  • Labeling of Proteins in vitro (P9302)


    1. Dissolve the vial of CoA substrate (50 nmol) in 50 μl of fresh DMSO to yield a labeling stock solution of 1 mM CoA substrate. Mix by vortexing for 10 minutes until all the CoA 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:
      Component Volume Final
      Deionized Water 29.25μl  
      1 M HEPES 2.5 μl 50 mM
      50 mM MgCl2 10 μl 10 mM
      40 µM SEP
      1.25 μl 1 µM
      50 µM ACP-tag or MCP-tag Purified Protein 5μl 5 µM
      250 µM CoA
      2μl 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 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 separated from the labeled ACP-tag or MCP-tag fusion protein by gel filtration or dialysis. Please refer to the vendor’s instructions for the separation tools used.