Abstract: 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.

Abstract: The present invention concerns 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 the 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 the ligand; and b) analyzing said sample for the presence of soluble or native target protein using two or more affinity reagents capable of binding to said soluble or native target protein with a higher affinity than to an unfolded and/or insoluble form of said target protein. The invention particularly concerns the use of two affinity reagents (e.g. antibodies) which are capable of distinguishing between soluble or native, and unfolded and/or insoluble forms of a target protein and whose detection e.g. by FRET based technology, allows the performance of the method without a separation step.