We know about the amount of impurities in agarose like sulfate content increase elelctroendosmosis. Is it known that different buffers used can influence the phenominon of EEO due to different cations, each molecule of which carry a specific number of water molecules with it. Can anybody have information about cations present in electrophoresis producing more EEO and which cations produce less contribution to EEO ?
Hello Ramesh, I do not think the problem with electro-endosmosis (EEO) is with the nature of cation. If you follow the mechanism of endosmosis properly, you will find that the major driving factors include the buffer, nature of the electrophoretic support medium and nature of protein involved. The choice of the electrophoresis buffer is one of the most important influences on the resolution of the electrophoretic system. This is because the buffer defines the pH which in turn determines the net charge, speed and direction of migration of the protein. Some electrophoretic support media adsorb hydroxyl ions from the buffer and create fixed (immobile) negative charges. This is the property responsible for endosmosis - the flow of solvent in the electric field during electrophoresis. The fixed charges are surrounded by a cloud of mobile positive ions such that when a current is applied, the mobile positive ions freely migrate to the cathode but the immobilized negative ions stay with the support material. Because the migrating positive ions are hydrated, they carry with them a shell of solvent molecules. The movement of positive ions and solvent towards the cathode is what creates the endosmotic effect during electrophoresis.Proteins that migrate towards the cathode must move against this force. Most proteins at the usual pH of electrophoretic buffers have sufficient negative charges to overcome this force however, proteins with few negative ions like gamma-globulin often remain still during electrophoresis. Support media like paper, cellulose acetate and conventional agarose contain fixed negative charges and so have pronounced endosmotic effect. Support systems like polyacrylamide or purified agarose gels have little endosmotic effect. Please follow the link belowArticle The effects of electroendosmosis in agarose electrophoresis
The EEO originates from the composition of the capillary wall, or supporting medium like gel, and its interaction with the electrophoresis buffer.
Why don't you start with using different types of agarose with more or less impurities. See https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis
This article give also information about electrophoresis buffers to be used.
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