Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources.
Purification and chemical characterization of proteins may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60% and 100% were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60% and 79%. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patterns, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.
Jungblut
P
P
Institut für Humangenetik, Freie Universität Berlin, Federal Republic of Germany.
Eckerskorn
C
C
Lottspeich
F
F
Klose
J
J
D000818:Animals; D001322:Autoanalysis; D015415:Biomarkers; D001923:Brain Chemistry; D002021:Buffers; D015180:Electrophoresis, Gel, Two-Dimensional; D051379:Mice; D008811:Mice, Inbred DBA; D011506:Proteins / Q000032:analysis*; D012995:Solubility
D015415:Biomarkers; D002021:Buffers; D011506:Proteins