Protein purification procedures are usually carried out at low temperature (0 - 4ºC) since most proteins are stable at low temperature. As the temperature increases from 0ºC to 37 - 40ºC their stability decreases significantly. Above 40ºC or so, most proteins become increasingly unstable and denature, and at neutral pH, the denatured proteins usually precipitate.

Individual proteins differ in their heat sensitivity and so this can be used for purification purposes. The temperature stability of the desired enzyme is determined by a trial experiment following enzyme activity in the cell extract after incubation at different temperatures for a set period of time. The minimum temperature at which gross inactivation occurs is noted. Once this temperature is known, less stable proteins can be preferentially inactivated by incubating the cell extract at a temperature 5 - 10ºC below this value for 15 - 30 min. The denatured precipitated protein is then removed by centrifugation.

It should be noted that the heat stability of the desired enzyme may be increased during heat denaturation by the presence of its substrate, product or a competitive inhibitor which bind to the active site and help stabilize the protein conformation. Since denaturation of cell proteins occurs to some extent at all temperatures and simply increases with increasing temperature, the total activity of the desired enzyme usually falls to some extent after a heat denaturation step. This procedure is therefore rather crude. However, it may be a useful early step for the purification of rather more heat-stable proteins.