Native gelelectrophoresis uses native or non-denaturing gels for the analysis of proteins in their folded, native state. The electrophoretic mobility of an intact protein is depended on its charge-to-mass ratio as well as on its physical shape and size. Chemically, proteins are structurally very complex and functionally sophisticated molecules that are known to man. The nature and position of each amino acid located within the long string of amino acid sequence that forms a protein determines its three-dimensional shape and function and therefore its migration behavior in a gel during an electrophoresis experiment.
Blue native PAGE or BN-PAGE
Blue native PAGE (BN-PAGE) is a native polyacrylamide gel electrophoresis technique in which the dye Coomassie Brilliant Blue provides the necessary charges to the protein complexes needed for electrophoretic separation so that they migrate into the gel employed. However, because Coomassie Blue binds to proteins it can act like a detergent which can cause protein complexes to dissociate <http://en.wikipedia.org/wiki/Dissociate>. In addition Coomassie Blue also potentially can quench the chemoluminescence or fluorescence of proteins containing prosthetic groups such as heme, chlorophyll or fluorescent dyes.
Clear native PAGE
Clear native PAGE (CN-PAGE) or Native PAGE allows separation of acidic water-soluble and membrane proteins using a polyacrylamide gradient gel. No charged dye is used here therefore the electrophoretic mobility of proteins in CN-PAGE is related to the intrinsic charge and shape of the proteins. The observed migration distance is depended on the protein charge, its size and the pore size of the gel. In general this method has a lower resolution than BN-PAGE but may offer advantages whenever the Coomassie dye will interfere with further analytical techniques. Furthermore, CN-PAGE is milder than BN-PAGE so it can retain labile supramolecular assemblies of membrane protein complexes that are dissociated under the conditions of BN-PAGE. However, to establish the best experimental conditions several trial runs maybe needed before the best separation conditions are found.
Quantitative preparative native continuous PAGE
Preparative native continuous gel electrophoresis (QPNC-PAGE) can be used to separate native, correctly folded protein complexes of interest. To be successful, the proper buffer system and apparatus will need to be selected or designed. For example, QPNC-PAGE can be used to identify and quantify metal cofactors comigrating with proteins in specific fractions. In addition, metalloproteins can be isolated and elucidated with the help of non-denaturing methods. Over the years many different gelelectrophoresis systems have been designed and are offer now by many commercial companies.
Blue native PAGE or BN-PAGE
Blue native PAGE (BN-PAGE) is a native polyacrylamide gel electrophoresis technique in which the dye Coomassie Brilliant Blue provides the necessary charges to the protein complexes needed for electrophoretic separation so that they migrate into the gel employed. However, because Coomassie Blue binds to proteins it can act like a detergent which can cause protein complexes to dissociate <http://en.wikipedia.org/wiki/Dissociate>. In addition Coomassie Blue also potentially can quench the chemoluminescence or fluorescence of proteins containing prosthetic groups such as heme, chlorophyll or fluorescent dyes.
Clear native PAGE
Clear native PAGE (CN-PAGE) or Native PAGE allows separation of acidic water-soluble and membrane proteins using a polyacrylamide gradient gel. No charged dye is used here therefore the electrophoretic mobility of proteins in CN-PAGE is related to the intrinsic charge and shape of the proteins. The observed migration distance is depended on the protein charge, its size and the pore size of the gel. In general this method has a lower resolution than BN-PAGE but may offer advantages whenever the Coomassie dye will interfere with further analytical techniques. Furthermore, CN-PAGE is milder than BN-PAGE so it can retain labile supramolecular assemblies of membrane protein complexes that are dissociated under the conditions of BN-PAGE. However, to establish the best experimental conditions several trial runs maybe needed before the best separation conditions are found.
Quantitative preparative native continuous PAGE
Preparative native continuous gel electrophoresis (QPNC-PAGE) can be used to separate native, correctly folded protein complexes of interest. To be successful, the proper buffer system and apparatus will need to be selected or designed. For example, QPNC-PAGE can be used to identify and quantify metal cofactors comigrating with proteins in specific fractions. In addition, metalloproteins can be isolated and elucidated with the help of non-denaturing methods. Over the years many different gelelectrophoresis systems have been designed and are offer now by many commercial companies.