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 , in the native , ie folded be resolved by the grating structure of a polyacrylamide gel proteins in an electric field.
Gel electrophoresis under native conditions is about preserving the physiological properties of the proteins to be separated. This could be for several reasons, for example
- to separate proteins that cannot be separated by other electrophoresis methods, such as phosphorylated and non-phosphorylated variants of the same protein
- to show biologically relevant conformations , such as proteins, which can occur as monomers , dimers, trimers, tetramers etc.
- to detect complex formation or protein-protein interaction of different proteins
execution
In contrast to SDS-PAGE , in which proteins are completely denatured in the presence of SDS under the supply of heat and a high concentration of negative charges is attached to them, gel electrophoresis under native conditions is exclusively dependent on the existing charges of the protein. Therefore, the choice of depends buffer system from the isoelectric point of the protein from. A protein with a pI value of 6.2 is weakly negatively charged in a buffer with pH 7 and therefore migrates more slowly to the anode in the electric field than another protein of pI 5. Strongly basic proteins (with pI values above 8) can be separated in pH-neutral buffers by exchanging the anode and cathode.
In addition to the charge of the proteins, their size and shape also influence the migration properties. In order to minimize these factors, polyacrylamide gels of low concentration and crosslinking are often used , which on the other hand reduces their mechanical stability and makes handling difficult.
In order to keep the proteins to be separated folded, the buffer must be adapted to the requirements of the proteins (reducing agent, etc.) and, if necessary, cofactors ( nucleotides , cations, etc.), which are required for stability and activity, must be added to the buffer or even in the gel will be given.
In order to remove any interfering substances that may be added during the curing reaction of the polyacrylamide gel, and to allow the ions of the running buffer to migrate, the gel can be run for a while without the sample to be separated before the separation run, and then the Renew run buffers ( pre-run gels ).
Some proteins require a low concentration of reducing compounds to protect them , such as dithioerythritol or mercaptoethanol . Such compounds can either interfere with the curing reaction of the polyacrylamide gel or even be destroyed during the curing reaction of the polyacrylamide gel. It would take a long time for electrically neutral molecules to diffuse into the gel. Therefore, instead of the otherwise usual electrically neutral reducing compounds, the amino acid cysteine, which also reduces disulfide bridges, can be electrophoretically immigrated. The isoelectric point of cysteine is at pH 5.02. At this pH value, cysteine cannot migrate into the gel.
In many of the buffer systems required for native gel electrophoresis, strongly basic and strongly reducing compounds are formed on the cathode , and strongly acidic and strongly oxidizing compounds, such as hypochlorites , are formed on the anode . To protect the sample from this, one should use several electrode buffer vessels , which are connected by power keys filled with electrode buffer, as shown in the upper part of the picture, and at least check the pH value of these buffer vessels regularly.
Since heat is generated during gel electrophoresis and since the amount of heat depends on the buffer system, the work is usually carried out under cooling; occasionally the exact electrophoresis conditions are also carefully tested.
literature
- Hermann Schägger, Gebhard von Jagow: Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. In: Analytical Biochemistry . Vol. 199, No. 2, 1991, pp. 223-231, PMID 1812789 , doi : 10.1016 / 0003-2697 (91) 90094-A .
- Hermann Schägger, WA Cramer, Gebhard von Jagow: Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. In: Analytical Biochemistry. Vol. 217, No. 2, 1994, pp. 220-230, PMID 8203750 , doi : 10.1006 / abio.1994.1112 .
- Michael Niepmann, Junfeng Zheng: Discontinuous native protein gel electrophoresis. In: Electrophoresis. Vol. 27, No. 20, 2006, ISSN 0173-0835 , pp. 3949-3951, PMID 16991206 , doi : 10.1002 / elps.200600172 .