Application Note CHEM001:Applications of Site-specific Biotinylated Synthetic Chemokines


Application Note CHEM001: Applications of Site-specific Biotinylated Synthetic Chemokines:

Ibt now offers a range of site-specifically biotinylated synthetic chemokines. Using the advantages offered by modern synthetic chemistry methods, a single biotin molecule is added to a specific location in the chemokine sequence. There are many applications of such reagents – some are detailed below.


Site-specific Biotinylation

Standard biotinylation techniques often result in a random, multiple incorporation of biotin, affecting biological activity. ibt has developped a biotinylation technique, that permits a selective biotinylation of growth factors to yield fully bioactive ligands (see application note IGF004). An alternative technique is chemical synthesis that allows the site specific biotinylation. Biotin is incorporated as part of the synthetic process giving total control over the site of labelling. This was illustrated by in a programme to identify antibodies against MCP-1 (5). The authors found that synthetic, site-specifically labelled MCP-1 was fully active, whilst the randomly multiply labelled recombinant material had only partial activity in a calcium flux assay. Additionally, the control offered by synthetic chemistry allows the role of biotinylation site to be investigated, as for example with biotinylated RANTES (10).

A research on the available on-line literature shows, that biotinylated chemokines may be used for a broad range of techniques. Biotinylated chemokines are used in flow cytometry to determine receptor density (1, 6, 8, 9, 12, 13, 19, 21). This technique was applied to the screening of potential therapeutic agents. Biotinylated MIP-1α and MIP-1β were used as reagents in assays to determine the effectiveness of a prototype antiviral compound (4).

Microscopy-based methods have been used to determine the presence of receptors for MCP-1 and MIP-1α on brain microvessels(2) and for MCP-1 and MIP-1α receptors on astrocytes (3) and for MIP-3 beta on HEK293 cells (9)

Blotting techniques including ligand blotting have been used for internalization and proteolysis studies (10, 11, 16, 17, 18). As an alternative technique for proteolysis studies ESI MS has been used (17). In addition biotinylated chemokines have been used in ELISA (14) and histochemistry (15). Surface plasmon resonance assay has been used to identify chemokine binding proteins (20).

The conclusion is, that biotinylated chemokines are valuable tools and may find new applications e.g. as described for our biotinylated IGF´s (see application note IGF004).


1. Dürig J., de Wynter E.A., Kasper C, Cross M.A., Chang J., Testa N.G., Heyworth C.M. “Expression of macrophage inflammatory protein-1 receptors in human CD34(+) hematopoietic cells and their modulation by tumor necrosis factor-alpha and interferongamma” Blood, 1998, Nov 1;92(9):3073-81.


2. Andjelkovic, A. V. Spencer, D. D., and Pachter, J. S. “Visualization of Chemokine Binding Sites on Human Brain Microvessels”. J. Cell Biol., 1999 145 (2), 403-412.


3. Andjelkovic AV, Kerkovich D, Shanley J, Pulliam L, Pachter JS. “Expression of binding sites for beta chemokines on human astrocytes,” Glia 1999 28(3), 225-35.

Lin to Abstract:


4. Schols D, Struyf S, Van Damme J, Esté JA, Henson G, De Clercq E. “Inhibition of T-tropic HIV strains by selective antagonization of

the chemokine receptor CXCR4” J. Exp. Med. 1997, 186(8), 1383-8.


5. Kruszynski M, Tsui P, Stowell N, Luo J, Nemeth JF, Das AM, Sweet R, Heavner GA. “Synthetic, site-specific biotinylated analogs of human MCP-1.” J. Pept. Sci. 2006, 12(5), 354-60.

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6.) Abonyo et al: Autoregulation of CCL26 synthesis and secretion in A549 cells: A possible mechanism by which alveolar epithelial cells regulate airway inflammtion. Am J Lung Cell Physiol Mol Physiol (1995)


7.) Fulkerson et al: CXCL9 inhibits eosinophil responses by a CCR3- and Rac2-dependent mechanism. Blood, 15 July 2005, Vol. 106, No. 2, pp. 436-443.


8) Kellermann, S. and McEvoy, L. M.: The Peyer’s Patch Microenvironment Suppresses T Cell Responses to Chemokines and Other Stimuli.

The Journal of Immunology, 2001, 167: 682-690.


9.)Otero et al: Opposite Fate of Endocytosed CCR7 and Its Ligands: Recycling versus Degradation. The Journal of Immunology, 2006, 177: 2314-2323.


10) Charnaux et al: RANTES (CCL5) induces a CCR5-dependent accelerated shedding of syndecan-1 (CD138) and syndecan-4 from HeLa cells and forms complexes with the shed ectodomains of these proteoglycans as well as with those of CD44. Glycobiology, 2005, vol. 15 no. 2, 119-130.


11.) Bodaghi et al: Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells.

J Exp Med. 1998 Sep 7;188(5):855-66.


12.) Bäckhed et al:Helicobacter pylori Infection Induces Interleukin-8 Receptor Expression in the Human Gastric Epithelium. Infect Immun. 2003 June; 71(6): 3357–3360.


13.) Addison et al: Overexpression of the duffy antigen receptor for chemokines (DARC) by NSCLC tumor cells results in increased tumor necrosis. BMC Cancer 2004, 4:28doi:10.1186/1471-2407-4-28



14.) Cooper et al:  RANTES in onchocerciasis: changes with ivermectin treatment. Clinical & Experimental Immunology, Volume 106 Issue 3, Pages 462 – 467.


15.) Nagaoka et al: Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon γ-inducible Protein 10 kDa (IP-10), during Early Gestation. The Journal of Biological Chemistry, 2003, 278, 29048-29056.


16.) Sun et al: CD26/dipeptidyl peptidase IV regulates prostate cancer metastasis by degrading SDF-1/CXCL12. Clin Exp Metastasis. 2008;25(7):765-76.

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17.) Ravi et al: Elastase Release by Transmigrating Neutrophils Deactivates Endothelial-bound SDF-1 and Attenuates Subsequent T Lymphocyte Transendothelial Migration. JEM 2004, Volume 200, Number 6, 713-724.


18.) Sadir et al: Heparan Sulfate/Heparin Oligosaccharides Protect Stromal Cell-derived Factor-1 (SDF-1)/CXCL12 against Proteolysis Induced by CD26/Dipeptidyl Peptidase IV. The Journal of Biological Chemistry 2004, 279, 43854-43860.


19.) Dagan-Berger et al: Role of CXCR3 carboxyl terminus and third intracellular loop in receptor-mediated migration, adhesion and internalization in response to CXCL11. Blood, 15 May 2006, Vol. 107, No. 10, pp. 3821-3831.


20) Martin et al: Chemokine Binding Protein M3 Prevents Diabetes Induced by Multiple Low Doses of Streptozotocin. The Journal of Immunology, 2007, 178: 4623-4631.


21.) Sutton et al.: Stromal Cell–Derived Factor-1/Chemokine (C-X-C Motif) Ligand 12 Stimulates Human Hepatoma Cell Growth, Migration, and Invasion. Mol Cancer Res January 1, 2007 5, 2.