Method of electrophoresis
View Patent ↗The present invention relates a method of electrophoretic separation of protein and/or peptide components of a sample in a convection stabilized medium. More specifically, the method comprises the steps to contact the sample with the separation medium; to apply a voltage across said medium; and to observe the results by analysis of one or more sections of the separation medium. In the present method, a disulphide-comprising compound is added before or during the procedure to make an excess of reactive disulphide groups accessible to react with the cysteine groups of the proteins and/or peptides all through the separation procedure. The present invention also relates to electrophoretic separation media that comprises reactive disulphide groups, such as polyacrylamide gels, and the use of a solution that comprises reactive disulphide groups to pretreat an electrophoretic separation medium.
1. A method of electrophoretic separation of one or more protein and/or peptide components of a sample, which method comprises
(a) contacting the sample with a convection stabilised separation medium;
(b) applying a voltage across said medium; and
(c) observing the results of the separation obtained by analysis of one or more sections of the separation medium;
wherein a disulphide-containing compound selected from the group consisting of bis-(2-hydroxyethyl) disulphide; bis-(2-hydroxypropyl) disuiphide; 3,3-dipropionamidedisulphide and 2,2′-dipyridyl disulphide is added to the separation medium or to the sample before step (a) in an amount sufficient to provide an excess of reactive disulphide groups during the separation and to prevent protein agglutination and streaking.
2. The method of claim 1 , wherein the separation medium includes one or more surfaces or spaces of capillary dimensions.
3. The method of claim 1 , wherein the separation medium comprises a gel.
4. The method of claim 1 , wherein one or more buffers are added to keep the pH and ionic strength essentially constant in the separation medium during the electrophoresis.
5. The method of claim 4 , wherein the sample is treated with a charged surfactant to mask the charge of the proteins and/or polypeptides therein before it is contacted with the separation medium and wherein the proteins and/or peptides are separated according to their molecular weight.
6. The method of claim 1 , wherein a stationary pH-gradient is formed by providing charged or chargeable groups along all of the separation distance in the convection stabilized separation medium and the proteins and/or polypeptides are separated according to their isoelectric points.
7. The method of claim 6 , wherein the charged or chargeable groups are non-mobile and affixed to or in a matrix.
8. The method of claim 7 , wherein the matrix is the separation medium.
9. The method of claim 6 , wherein the separation medium includes carrier ampholytes.
10. The method of claim 9 , performed in an apparatus including at least two chambers separated from each other by at least one membrane.
11. The method of claim 1 , wherein a gradient of pH and/or ionic strength is formed by providing charged or chargeable groups along a part of the separation distance in the convection stabilized separation medium and wherein the proteins and/or polypeptides are separated according to their respective transport velocities.
12. The method of claim 11 , wherein the separation medium includes carrier ampholytes.