Abstract
Blue native electrophoresis is one of the most popular techniques for mass estimation of native membrane proteins, but the use of non-optimal mass markers and acrylamide gels can compromise accuracy and reliability of the results. We present short protocols taking 10-30 min to prepare optimal sets of membrane protein markers from chicken, rat, mouse, and bovine heart. Especially heart materials from local supermarkets or butcher's shops, e.g. chicken or bovine heart, are ideal sources of high mass membrane protein standards. Considerable discrepancies between the migration behavior of membrane and soluble markers suggest using membrane protein markers for mass estimation of membrane proteins. Soluble standard proteins can be used, with some limitations, when soluble proteins are the focus. Principles and general rules for the determination of mass and oligomeric state of native membrane and soluble proteins are elaborated, and potential pitfalls are discussed.
Highlights
Blue native electrophoresis is one of the most popular techniques for mass estimation of native membrane proteins, but the use of non-optimal mass markers and acrylamide gels can compromise accuracy and reliability of the results
Dodecylmaltoside-solubilized complexes from bovine heart mitochondria and soluble proteins of the high molecular weight kit were used as mass markers
Mass estimation by Clear native electrophoresis (CNE) is essentially restricted to acidic water-soluble proteins in many cases (Fig. 5D) because no charge shift is imposed on the native proteins, and most membrane proteins are resolved insufficiently
Summary
Electrophoretic Techniques—BNE, CNE, hrCNE, and associated techniques like two-dimensional Tricine-SDS-PAGE and staining were performed as described [3,4,5]. It is important to note that, at the end of gradient mixing, a certain volume of the acrylamide solution remains in the gradient mixer and in the tubing This dead volume must be measured at least once to find the actual final acrylamide concentration in the gradient gels. Mitochondrial membrane protein complexes and supercomplexes were well preserved using frozen and non-frozen heart as analyzed by two-dimensional BN/SDS-PAGE under various detergent conditions (not shown). Solubilization of Membrane Protein Complexes from Chicken and Mammalian Heart for Mass Estimation by Native Electrophoresis—It is important to use the same detergent that was used for solubilization of the protein of interest to solubilize the mitochondrial mass markers.
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