Fundamental Studies on Paper Electrophoresis of Human Serum Protein

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1. To make the voltage between both ends of the horizontal portion of paper as equal as possible in each experiment, it is necessary to keep the tensions of paper constant and to maintain the distances from the surface of buffer solution to the horizontal portion of paper constant, too.2. After electrophoresis, pH-values of barbiturate buffer solution deviate from the original pH, to some extent, in both of the anode and cathode containing vessels. They deviate in the paper ends containing vessels only when the applied voltage is relatively high or/and electrophoresis hour is extremely long. pH-Values of total buffer solutions, however, have little change if they are mixed and dissolved well after electrophoresis. Therefore, the same buffer solution can be used three or four times repeatedly, although some changes of ionic strength and some contaminations with paper fibres and bacilli may occur.3. At higher voltages, 1) the migration velocities are large, but the electrophoretic patterns are worse separated, especiaily when electrophoresis hour is short. 2) only in long electrophoresis hours, they are well separated and less blurred in their zones because of the enhancement of ionic strength on paper. 3) the length of Alb developped from γ-gl is short because both of the migration velocities and the buffer flow augment.4. When the ionic strength of buffer solution is stronger, 1) the migration velocities are small, 2) the electrophoretic patterns are well separated and their zones are less blurred because the zonal diffusion is restricted by the augmented ion transportation. 3) α1-gl is well separated from Alb. 4) globulins, especially, γ-gl displace to the more acidic part on paper if the migration distances of BPB stained Alb is fixed to a definite from the application point.5. The migration distance of BPB stained Alb from the application point is a good index for checking the electrophoretic separation of serum protein, because the electrophoresis hour required for the good separation is not definite but dependent on the experimental temperature and so on.6. In the author's laboratory, serum protein is submitted to the electrophoretic run usually at the constant voltage of 200V, using sodium barbiturate buffer of pH 8.6 and ionic strength of 0.075. When serum volumes of 0.01ml/2cm (width of a paper strip) or less are applied, BPB stained Alb is allowed to migrate 8cm from the application points. When serum volumes of 0.02ml or more, BPB stained Alb is allowed to 12cm, likewise. If the electrophoretic patterns are poorly separated and blurred, for example, owing to therelative low temperature, voltage and electrophpresis hour (expressed as the migration distance of BPB stained Al), for the first choice, and nextly the ionic strength are adjusted so as to ameliorate the results described in 3 and 4.