Introduction
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.
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.
Materials
- Capture Substance:
- Solubilized CCR7 receptor - CCR7-EMP [Abilita Bio]
- Purified to homogeneity
- Stock concentration: 2 mg/ml in Buffer: 20 mM HEPES pH 7.2, 500 mM NaCl, 0.05 % DDM, 0.01 % CHS
- Solubilized CCR7 receptor - CCR7-EMP [Abilita Bio]
- Detection Substance:
- 10 µg CCL19-biotin [ChemoTactics, B-CCL19-10µg]
- Reconstituted in 9 µl PBS (approximately 100 µM)
- 10 µg CCL19-biotin [ChemoTactics, B-CCL19-10µg]
- Negative Control:
- Control GPCR-EMP [Abilita Bio]
- Purified to homogeneity
- Stock concentration: 1.5 mg/ml; Buffer: 20 mM HEPES pH 7.2, 500 mM NaCl, 0.05 % DDM, 0.01 % CHS
- Control GPCR-EMP [Abilita Bio]
- Enzyme:
- Streptavidin-HRP [Pierce, Cat No. 21130]
- Substrate Solution:
- Tetramethylbenzidine (TMB) Substrate Solution [Sigma, Cat. No. T0440]
- Coating Buffer: Phosphate-buffered saline pH 7.4 [Sigma, Cat. No. 79383]
- Wash Buffer: 20 mM HEPES pH 7.0, 150 mM NaCl, 0.05 % DDM/CHS
- Binding Buffer: 20 mM HEPES pH 7.2, 150 mM NaCl, 0.05 % DDM/0.01 % CHS + 1% BSA
- Acquisition Instrument: Molecular Devices SpectraMax i3 plate reader
- 96-Well Nickel Coated Plates: [Thermo/Pierce, Cat. No. 15442]
Protocol
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Example Data & Analysis of Results
Results were analyzed by Prism using one-site binding model to calculate KD = 72 nM.
Figure D. Saturation curve measuring B-CCL19 binding to immobilized CCR7-EMP on nickel coated plate.