CN-PAGE

The Colorless Native Polyacrylamide Gel Electrophoresis (CN -PAGE, in German, colorless native polyacrylamide gel electrophoresis ') is a variant of the native gel electrophoresis, are separated by the lattice structure of a polyacrylamide gel in the native, ie folded proteins in an electric field.

In gel electrophoresis under native conditions, it is necessary to obtain the physiological properties of the fractionated proteins. This can have several reasons, for example,

• Proteins to unravel, which can not be separated with other electrophoretic methods, such as phosphorylated and non-phosphorylated variants of the same protein
• Biologically relevant conformations show like proteins which may act as monomers, dimers, trimers, tetramers, etc.
• In order to demonstrate complex formation or protein-protein interaction of various proteins

Implementation

Unlike SDS-PAGE, the proteins are fully denatured at in the presence of SDS under heat and a very high concentration of negative charge is attached to the gel electrophoresis under native conditions is dependent exclusively on the current loads of the protein. Therefore, the selection of the buffer system from the isoelectric point of the protein depends. A protein with a pI value of 6.2 is loaded weakly negative in a buffer with pH 7 and therefore migrates in the electric field towards the anode slower than another protein of pI 5 Strongly basic proteins ( with pI values ​​above 8 ) can be separated into neutral pH buffers, by interchanging the anode and cathode.

In addition to the charge of the proteins and their size and shape affect the migrant characteristics. To minimize these factors, one commonly used polyacrylamide gels of low concentration and cross-linking, which in turn reduces its mechanical strength and makes handling more difficult.

To hold the folded proteins to be separated, the buffer to the requirements of the proteins need to be adjusted ( reducing agent, etc.) and, optionally, co-factors (nucleotides, cations, etc.) which are required for stability and activity, with the buffer, or even the gel are given.

To remove the added during the curing of the polyacrylamide gel and the resulting, possibly interfering substances, and to let immigrate to the ions of the running buffer, you can run the gel for a while before separating run run electrically without the sample to be separated, and then the running buffer renew (English pre -run gels ).

Some proteins need to protect low concentrations of reducing compounds, such as, for example, dithioerythritol or mercaptoethanol. Such compounds may interfere with the curing of the polyacrylamide gel either, or even in the curing of the polyacrylamide gel are destroyed. The diffusing into electrically neutral molecules in the gel would last very long. Therefore, one can be in place of the usual electrically neutral reducing compounds which are also disulfide -reducing amino acid cysteine ​​migrate electrophoretically. The isoelectric point of cysteine ​​is at a pH of 5.02. At this pH, cysteine ​​can not migrate into the gel.

In many buffer systems required for native gel electrophoresis produced at the cathode strongly basic and strongly reducing compounds, and at the anode strongly acidic and strongly oxidizing compounds, such as hypochlorites. To protect the sample against one should use several electrode buffer vessels, which are connected by the buffer -filled electrode current key, as shown in the upper part of the image, and to check at least the pH of the buffer tubes at regular intervals.

Since the gel electrophoresis of heat is generated, and since the amount of heat depends on the buffer system is usually carried out under cooling, sometimes the exact electrophoresis also be carefully tested.