Protein Blotting by the Capillary Method

Abstract

The ability to transfer (blot) separated proteins from a polyacrylamide gel matrix onto a sheet of nitrocellullose paper (where the proteins bind to the surface of the paper) has provided a powerful tool for protein analysis. Once immobilized on the surface of the nitrocellulose sheet, a variety of analytical procedures may be carried out on the proteins that otherwise would have proven difficult or impossible in the gel. Such procedures may include hybridization with labeled DNA or RNA probes, detection with antibodies (probably the most commonly used procedure), detection by specific staining procedures, autoradiographic assay, and so forth. The most commonly used, and indeed most efficient, methods for transferring proteins from gels to nitrocellulose (blotting) are by electrophoresis, and these methods are described in Chapters 39 and 40. An alternative method, capillary blotting, is described here. Although this method takes longer than electroblotting methods (it takes overnight) and transfer of proteins from the gel is not as complete as it is for electroblotting (although sufficient protein is transferred for most purposes) the method does have its uses. It is of course ideal for those who only wish to carry out occasional blotting experiments and therefore do not wish to commit themselves to the purchase or the purpose-built apparatus (plus power pack) needed for electroblotting. Second, this method is particularly useful for blotting isoelectric focusing gels where the proteins are at their isoelectric point (i.e., they have zero overall change) and are not easy therefore to transfer by electrophoresis. The method simply involves placing the gel on filter paper soaked in buffer. Buffer is drawn through the gel by capillary action by placing a pad of dry absorbent material on top of the gel. As the buffer passes through the gel, it carries the protein bands with it, and these bind to the nitrocellulose sheet that is placed between the top of the gel and the dry absorbent material.