Protein stability assay

The method of any preceding claim, wherein steps i), ii) and iii) are each independently conducted at a substantially constant temperature.

The method of any preceding claim, wherein steps ii) and iii) are conducted at the same substantially constant temperature.

The method of any preceding claim, wherein steps i), ii) and iii) are conducted at the same substantially constant temperature.

The method of any preceding claim, wherein steps ii) and/or iii) of the method are conducted at ambient temperature.

The method of any preceding claim, wherein the assaying of step iii) detects the protein target in its folded state.

The method of any preceding claim, wherein the assaying is performed using:

• Homogeneous proximity assays or enzyme fragment complementation assay.

The method of any preceding claim, wherein the chemical denaturing agent comprises at least one of:

• an acid;
• an alcohol;
• and a ketone.

The method of any preceding claim, wherein the chemical denaturing agent comprises a C1-C5 acid.

The method of any preceding claim, wherein the chemical denaturing agent comprises a C2-C5 alcohol.

The method of any preceding claim, wherein the chemical denaturing agent comprises a C2-C5 ketone.

The method of any preceding claim, wherein the protein target is provided in step i) at a concentration of between 0.1 ng/μl and 5 μg/μl.

The method of any preceding claim, wherein the compound of interest is provided in step i) at a concentration of up to 100 mM.

The method of any preceding claim, wherein the protein target is exposed to the compound of interest for a period of at least 5 minutes.

The method of any preceding claim, wherein the protein target is exposed to the compound of interest for a period of between 30 minutes and 24 hours.

The method of any preceding claim, wherein the protein target is provided in the denaturing mixture at a concentration of between 0.1 ng/μl and 5 μg/μl.

The method of any preceding claim, wherein the protein target and chemical denaturing agent are contacted with one another for a period of at least 5 minutes.

The method of any preceding claim, wherein the protein target and chemical denaturing agent are contacted with one another for a period of between 30 minutes and 24 hours.

The method of any preceding claim, wherein the protein target is part of a complex with an associated protein.

The method of any preceding claim, wherein the proportion of the protein target in its folded state and/or the proportion of protein in its denatured state is determined directly by measurement of the target protein.

A method of identifying whether a protein target and a compound of interest bind to one another, the method comprising, in order, the steps of:

• i) exposing the protein target, in the presence of an associated protein that is part of a complex with the protein target, to the compound of interest for a time sufficient to allow binding of the compound of interest to the protein target;
• ii) contacting the associated protein with a chemical denaturing agent to produce a denaturing mixture;
• iii) assaying the products of the denaturing mixture to determine the proportion of the associated protein in its folded state and/or the proportion of the associated protein in its denatured state, wherein a change in the proportion of the associated protein in its folded state as compared to a suitable control indicates that the protein target and compound of interest bind to one another, and wherein steps ii) of the method is conducted at a substantially constant temperature.