Methods for determining ligand binding to a target protein using a thermal shift assay

摘要

The invention is directed to a method of determining whether a non-purified sample contains a target protein bound to a ligand of interest comprising the steps of: a) exposing said non-purified sample to a temperature which is capable of causing or enhancing precipitation of the unbound target protein to a greater extent than it is capable of causing or enhancing precipitation of the target protein bound to said ligand; b) processing the product of step a) in order to separate soluble from insoluble protein; and c) analyzing either or both the soluble and insoluble protein fractions of step b) for the presence of target protein, wherein said target protein is not detected on the basis of enzymatic activity of a tag, peptide, polypeptide or protein fused thereto. Particularly, the invention may be used to determine whether drugs can bind to their protein targets in samples derived from patients to ascertain whether a certain drug can be used in a therapy for that patient. Additionally, the invention is directed to an instrument for use in the methods of the invention and use of a kit in the methods of the invention comprising an antibody and/or a non-protein fusion tag.

主权项

1. A method for identifying a ligand capable of binding to a target protein comprising the steps of:
a) exposing a non-purified sample comprising the target protein and a test molecule to a temperature which is capable of causing or enhancing precipitation of the unbound target protein to a greater extent than it is capable of causing or enhancing precipitation of the target protein bound to the ligand, wherein the target protein in said non-purified sample is comprised within or on cells; b) subjecting said sample to conditions capable of causing cell lysis; c) separating soluble from insoluble protein in the product of step b); and d) analysing the soluble protein fractions of step c) and optionally the insoluble protein fraction of step c) for the presence of the target protein, wherein the target protein is detected by affinity binding to a detection moiety or by mass spectrometry, and wherein the test molecule is identified as a ligand capable of binding to the target protein if the level of target protein in said soluble fraction is higher than the level of target protein detected in a soluble fraction obtained in a control reaction.