Is anyone out there purify your protein with heat treatment? I am dealing with thermostable fusion protein. I have done purification within two steps by using affinity chromatography methods and heat treatment. Heat treatment will induce the specific denaturation and precipitate of the E.coli proteins but not the thermostable proteins. I can get single band after heat treatment at 60C for 30 minutes. Lately, i try to increase the starting culture for purification. And after heat treatment with the same condition, i cannot get the same result anymore. There are two other bands (smaller size than 14.4kDa). Can i repeat again heat treatment to remove the purity bands?