Hancock Lab

Preparing biotinylated peptide LL37

Categorized: Antibiotics and Antimicrobial Peptides

Biotinylation of peptides at cysteine with Nalpha-(3-maleimidylpropionyl)biocytin.

Author:

Annett Rozek (Postdoctoral fellow, Hancock Lab); July 2005.
(Revised/updated by Susan Farmer, Research Associate, Hancock Lab, October 2012).

References:

  1. Lau, Y.E., A. Rozek, M.G. Scott, D.L. Goosney, D.J. Davidson and R.E.W. Hancock. 2005. Interaction and cellular localization of the human host defence peptide, LL-37, with lung epithelial cells. Infect. Immun.73:583-591.
  2. Bowdish, D.M.E., D.J. Davidson, Y.E. Lau, K. Lee, M.G. Scott, and R.E.W. Hancock. 2005. Impact of LL-37 on anti-infective immunity. J. Leukocyte Biol. 77:451-459.

The Nalpha-(3-maleimidylpropionyl)biocytin is from Molecular Probes/Invitrogen, cat# M-1602.

LL-37 needs to have a cysteine to link to the biocytin. At the time we had decided to add it at the C-terminus (“LL37C”).

Procedure for LL37C:

  1. Dissolve 12 mg of Nalpha-(3-maleimidylpropionyl)biocytin (MW=524 g/mol) in 20 ml of Tris buffer (50 mM, pH 7) at room temperature in 50 ml Falcon polypropylene tube. Put on shaker until completely dissolved (about 10-15 min).
  2. Dissolve 20 mg LL37C (MW=4596 g/mol) in 10 ml of same buffer. It should dissolve immediately.
  3. Combine reagents and let react for 2h on shaker at room temperature.
  4. Wrap tube with aluminum foil to protect from light.
  5. Stop reaction and scavenge excess Nalpha-(3-maleimidylpropionyl)biocytin by adding 10 ul 2-mercaptoethanol.
  6. Separate on reverse phase column (Resource RPC-3, Pharmacia) using a water (0.1% TFA)/acetonitrile (0.1% TFA) gradient. Biotinylated LL37 elutes around 40% acetonitrile, scavenged biocytin elutes around 20 %).

Yield is ~50%, 4.8 mg (quantified by LOWRY, BCA), 12.8 mg (by weight).

 

 

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