Recent evidence suggests that the human proteome probably numbers in the hundreds of thousands. A major tool for analyzing proteins is 2-D polyacrylamide gel electrophoresis (2-D PAGE). This procedure is tedious and time-consuming. It typically involves: (1) preparing a gel matrix with a specific pH gradient and performing the first dimensional isoelectric focusing (IEF) step; (2) equilibrating the IEF gel in the second dimension buffer, and (3) transferring the proteins from the IEF gel to the second dimension gel and running the second dimension separation, according to molecular weight.

The present invention provides for a high-speed, high-resolution 2-D method for separating proteins on high protein-binding membranes such as PVDF or nylons. The separation in both dimensions is performed on the same membrane which just has to be rotated in the electric field after the buffer change. The 2-D membrane electrophoresis is carried out at room temperature without cooling. No diffusion of the separated proteins was observed. Membranes of varying sizes (e.g. 8 cm x 8 cm to 20 cm x 20 cm or even larger) can be employed. When large membranes are used, separation of a thousand or more proteins can be accomplished quickly. Since electrophoresis is carried out directly on a protein-binding membrane, the subsequent immunodetection can be performed directly on that membrane. Among its many applications, this new method may find use in both expression proteomics such as finding markers for different cancers and functional proteomics such as studying protein-protein interactions.

The 2-D membrane electrophoresis method has several advantages over 2-D PAGE. (See Figure.) First, the entire 2-D membrane electrophoresis can be completed in about 20 minutes (vs. 1-2 days for 2-D PAGE). Second, the 2-D membrane electrophoresis is carried out under non-denaturing conditions, thus preserving enzymatic activities, protein-protein interactions, protein-ligand interactions, etc. Third, due to the organic solvent buffers used in 2-D membrane electrophoresis, both hydrophobic and hydrophilic proteins can be separated. Finally, unlike conventional IEF, this new method does not require ampholytes or preformed pH gradient strips, which are expensive and time-consuming to prepare. Proteins that are within 5 isoelectric points of the pH of the organic solvent buffers can be efficiently separated. Many inexpensive organic solvent buffers that cover the pH range from 3 to 10 were developed.

A new dye that stains proteins on membranes with sensitivity similar to that of colloidal gold (~ 2 ng) was also discovered in the course of this research. Both staining and destaining steps are completed in about 1-2 minutes each. Proteins bound to membranes that were previously reported to be difficult to stain (e.g. nylon membranes) are stained with the same sensitivity using this dye. Once stained, the bound dye can easily be removed.

File Number: 456FC 

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