I am using pEt28 and E. coli C43 to express a prokaryotic integral membrane protein and based on differential centrifugation and solubilization with detergent, the protein has been successfully inserted in the cytoplasmic membrane. FYI, I checked all fractions collected from differential centrifugation on Western Blot and the protein was only detected in the solubilized membrane fraction. I'm reading up about protein localization but find it difficult to understand how it is correctly inserted in the membrane instead of in the cytoplasm like most soluble proteins are. Thank you in advance.