Multiplexed 2D Western blot (2D mxWb) method. (A) Proteins from several samples are separated simultaneously with isoelectric focusing. (B) The strips are then successively loaded onto a polyacrylamide gel. The first strip is run for about 10 to 20 minutes; after this time, this strip is removed, the gel surface is rinsed with diiH2O, and the next strip is loaded and run for another 10 to 20 minutes; after this time, the procedure is repeated with the next strip until all of the strips are successively loaded in the SDS gel. (C) When all the strips have been run, the proteins are transferred onto a PVDF membrane and probed with specific antibodies. For the detection of ApoE, we used anti-goat ApoE antibody (CalBiochem) as the primary Ab and donkey anti-goat IgG-HRP as the secondary Ab.