Abstract
Sources of errors in quantitative paper electrophoresis of the serum proteins have been investigated, and found that the sources of errors are the so-called trail, difference of dye-combining capacity of each protein fractions, influence of the amount of protein on unit area of the paper to the optical density with white light, and the artifact of oiling process in densitometry. From the experimental results, to obtain reproducible data, following standard procedure is recommended.
1. For accurate quantitation, the electropherogram should be clear-cut, but the distance between each fractions should be reduced as narrow as possible to minimize the trail effect of the migrated protein. For this purpose, barbital buffer of higher ionic strength (0.1) than conventionally used (0.05 to 0.06) should be employed, and migration should be done with low voltage (75V/20cm).
2. Another measure to minimize the protein-trail effect is to select a filter paper such as Whatman No.1 or Toyo No.51 which adsorbs less protein.
3. To eliminate the densitometric artifact, preliminary removal of moisture and air in the stained paper and oil is absolutely necessary.
4. There are certain limitation of protein amount on unit area of the paper to obain linear relationship between protein amount and optical density with ordinary photocell-white light densitometer. In other words, optical density of dense area (e. g. center part of albumin) is not proportionally higher than that of light area (e. g. alpha-, beta-globulin part). To obtain Beer's relationship, the protein amount on the unit area should be below 0.0035mg/mm2. In practice, less than 0.01ml of serum diluted with buffer to the total protein concentration of 6.0g/dl should be applied evenly 3cm wide. For this reason, thinner paper is preferable, and it should be avoided to apply repeatedly even with a sample of low protein concentration, and when there is found a point which over 1.0 of optical density in densitometry, it should be re-migrated with less amount of the sample.
5. In the final quantitative analysis of the densitometric curves, the trail effect should be cut as described in the text.
In this experiment, in addition, I have confirmed following facts; that (1) trail is not only originate from albumin, but also from globulins, and its appearance is not additive, and there are certain point of saturation according to the property of paper and serum.(2) The amidoblack 10 B-combining capacity of alpha-globulin is almost same with that of albumin, but beta, and gamma-globulin combine slightly less than albumin.
