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Protein-ligand Binding Assay by Liquid Chromatography-Mass Spectrometry
Published: Aug 20, 2011 DOI: 10.21769/BioProtoc.124 Views: 16827
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Abstract
Protein-small molecule binding coefficients are determined by quantitative LC-MS in this method. Traditional biochemical plot is consequently used to set up the binding curve between a known protein and a small molecule (<1000 Da) to determine the affinity constant (Kd) and the stoichiometry (Bmax).
Keywords: Ligand binding
LC-MS
Molecular filtration
Protein interaction
Binding Curve
Materials and Reagents
- Purified protein (preferred in solution without any detergent like Tween, Triton, etc., concentration > 1 mM)
- Small molecule standard (best of highest available purity/grade, dissolved in the same solution used for proteins, in a 2-fold series of 10x desired concentration)
Note: To minimize organic solvent effect on binding, the stock solution has to be diluted in the same series in organic solvent first, and then mixed into the aqueous solution to make final standards. - Zeba spin desalting columns micro (Pierce Antibodies, catalog number: 89877 and 89878 )
- Methanol (mass spec grade)
Equipment
- Microcentrifuge
- Quantitative LC-MS (such as HPLC-coupled triple quad)
- Eppendorf Protein LoBind tubes (0.5 ml and 1.5 ml)
- LCQuan software (Thermo Fisher Scientific)
Procedure
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
Category
Systems Biology > Interactome > Protein-ligand interaction
Biochemistry > Protein > Interaction > Protein-ligand interaction
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