Chemokine Assays & Protocols
Biotinylated chemokines are very versatile in nature due to their ability to bind various streptavidin conjugates. Their research applications allow flexibility into many different assays. Here we highlight a few of these standard methods.
Flow Cytometry with Biotinylated Chemokines
A. Uptake of 20nM CXCL12-biotin by U937 cells in the presence (red trace) and absence (cyan) of a CXCR4 inhibitor, AMD 3100. U937 cells are not stained by streptavidin-PE in the absence of CXCL12-biotin (orange).
B. Uptake of 20nM CCL2-biotin by THP1 cells (dark green trace) is abolished by the addition of CCL7 (orange) but not CCL14 (light green), suggesting a CCR2-specific internalization. THP1 cells are not stained in the absence of CCL2-biotin (cyan).
B. Uptake of 20nM CCL2-biotin by THP1 cells (dark green trace) is abolished by the addition of CCL7 (orange) but not CCL14 (light green), suggesting a CCR2-specific internalization. THP1 cells are not stained in the absence of CCL2-biotin (cyan).
|
Activation of chemokine receptors by their cognate ligands leads to internalization of the receptor-chemokine complexes, which can be monitored by the use of fluorescent labeled ligands. This process can be exploited to detect the receptor expression and has been successfully used to identify the cells expressing the receptors of interest within a complex mixture of primary cells.
In this protocol, biotinylated CXCL12 is pre-mixed with phycoerythrin (PE)-conjugated streptavidin and subsequently incubated with U937 cells at 37°C in the presence or absence of AMD3100, a small-molecule inhibitor of its receptor, CXCR4. CXCL12 internalization is detected by flow cytometry, and its inhibition by AMD3100 suggests the receptor-mediated endocytosis. |
|
|
Biotinylated chemokines have a wide range of assay applications. Enzyme-Linked ImmunoSorbent Assay (ELISA) is a powerful technique that can measure the presence of a substance or chemokine with an antibody. Using different streptavidin conjugates, one can study chemokine internalization, receptor binding, or receptor expression levels. Their ability to recognize functional forms of receptors has been exploited by Abilita Bio, our collaborator, to demonstrate the correct folding of their recombinant stabilized receptor (EMP, or Enabled Membrane Protein) in an ELISA assay.
In this protocol, biotinylated CCL19 is used to measure the binding of a purified sample of stabilized CCR7 (CCR7-EMP) generated by Abilita Bio. By using streptavidin-HRP, one can construct the binding curve of CCL19 to CCR7-EMP and calculate the dissociation constant. 
|
|
Introduction
To test the migration ability of cells in response to the simulation by chemokine. The EC50 of each chemokine will depend on the cell line and receptor. For example, to test the migration of CXCR4 expressed in Jurkat cells in response to SDF-1a, use 0.003, 0.012, 0.048, 0.019, 0.75. 3.0 nM SDF-1a. Protocol
|
|
ChemoTactics Published Applications
|
Check out some recent publications that utilized our chemokine products to study chemokine interactions.
If you are interested in developing a new assay we offer consultation services as well to ensure your experimental design works with our products. Contact us for more info. |
|
Questions about our protocols? Contact us and we can help you.
