ISISBio:Protocols/Sortase mediated ligation/Small molecule ligation

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We have used Sortase to attach small molecule fluorophores to proteins but there is no reason why similar constructs shouldn't work for a very wide range of small molecule labels. Obvious possibilities include azides, alkynes, alkenes, biotin, and cysteine. We generally carry the reaction out overnight at room temperature and have not examined the effect of temperature extensively.

Design of the small molecule ligation partner

We have investigated the rate and yield of reaction for substrates of the form H-GGnK(Fluor)-OH which are straightforward to synthesise and relatively cheap to purchase from general peptide suppliers. We have observed no difference in rate of reaction or yield for n=1-3 and would therefore advise the use of substrates of the form H-GGK(Fluor)-OH on the grounds of ease and price. We have used fluorescein amine exclusively. Others have reported the use of similar constructs with three glycines and rhodamine but we see no reason why other fluorophores and other molecules should not work with two glycines. We recommend using as high a concentration of ligation partner as is feasible (or affordable) to obtain the highest yield.


  • Sortase A
  • LPETGG-tagged protein target
  • Small molecule construct
  • Sortase buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.5)

Standard Ligation Conditions

  • Target protein: 5 – 200 µM
  • Ligation partner: 100 µM – 1 mM (at least 20-fold excess over protein)
  • Sortase A: 50 nM


  1. Mix reactants together and incubate overnight at room temperature. Yes, it really is that simple (see here for an example).
  2. Run gel to confirm labeling reaction if appropriate
  3. Purify product


The small molecule ligation partner can be readily removed by gel filtration and residual Sortase can be removed if required by gel filtration (if the sizes are sufficiently different) or nickel affinity chromatography. Remember the desired product is in the flow through.

The yield of desired product generally follows the ratio of protein to ligation partner. Reducing the concentration of ligation partner will therefore reduce the yield. It also tends to slightly increase the amount of hydrolysis product observed. Increasing the Sortase concentration will speed up the reaction but will also increase the amount of hydrolysis product. Reactions are probably complete in 4-6 hours depending on target protein concentration but we have found overnight incubation convenient.


Relevant papers and books

  1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. PubMed ID:6947258 | HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. PubMed ID:13718526 | HubMed [Jacob-JMB-1961]
  3. Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. ISBN:0879697164 [Ptashne-Genetic-Switch]
All Medline abstracts: PubMed | HubMed


    • Cameron Neylon 11:45, 28 May 2008 (EDT):Who has experience with this protocol?