Preparing biotinylated peptide LL37
Categorized: Antibiotics and Antimicrobial Peptides
Biotinylation of peptides at cysteine with Nalpha-(3-maleimidylpropionyl)biocytin.
Annett Rozek (Postdoctoral fellow, Hancock Lab); July 2005.
(Revised/updated by Susan Farmer, Research Associate, Hancock Lab, October 2012).
- 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.
- 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:
- 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).
- Dissolve 20 mg LL37C (MW=4596 g/mol) in 10 ml of same buffer. It should dissolve immediately.
- Combine reagents and let react for 2h on shaker at room temperature.
- Wrap tube with aluminum foil to protect from light.
- Stop reaction and scavenge excess Nalpha-(3-maleimidylpropionyl)biocytin by adding 10 ul 2-mercaptoethanol.
- 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).
- Peptides (2)
- Antibiotics and Antimicrobial Peptides (6)
- Bacterial Genetics (22)
- Protein Purification and Gel Electrophoresis (17)
- Liposome Methods (6)
- Outer Membranes (26)
- Biochemical Assays (16)
- Microbiological Techniques (8)
- Host-Pathogen Interactions (3)
- Functional Genomics (1)
- Immunology Based Methods (8)