A simple, versatile, sensitive and volume-independent method for quantitative protein determination which is independent of other external influences.

A method is described which in principle is a quantitative "spot analysis". 0.5-5 microliter of a protein solution in the concentration range between 0.01-10 mg/ml is used for one determination. The sample is taken up by capillary attraction in a 0.5-, 1-, 2- or 5-microliters capillary and transferred to a cellulose acetate strip. The protein is fixed and stained simultaneously by dipping the cellulose acetate strip into a solution of Amido Black or a benzoxanthene derivative (Hoechst 2495) dissolved in in methanol/acetic acid. After elution of the excess of dye (3 x 5 min) the quantitative evaluation can be performed in different ways: 1) The sample is fixed and made transpartent by incubation in dioxane/1-butanol and evaluated densitometrically (Amido Black 10B) or 2) the evaluation is performed in situ by spot fluorometry (Hoechst 2495). 3 The sample can be dissolved together with the acetate layer completely in dioxane, dimethylsulfoxide or N,N-dimethylformamide and evaluated photometrically or 4) fluorometrically. 5) Highest sensitivity is reached if the fluorochrome (Hoechst 2495) bound to the protein is eluted with 15% NH4OH and measured fluorometrically. There is a linear correlation with a correlation coefficient of 0.999 between the fluorescence and a protein amount of 10 ng-20 micrograms. In addition to its simplicity, the method has the advantage of being independent of or well-defined against other external influences, e.g., sodium dodecyl sulfate, mercaptoethanol, Triton X-100, etc. The stainability of a protein with Amido Black is influenced stoichiometrically by sodium dodecyl sulfate (not by mercaptoethanol) whilst the staining with Hoechst 2495 is not at all affected. As there is linear correlation between the area of a spot on an acetate layer and the volume applied in the range between 0.5 and 5 microliters, (only influenced stoichiometrically by the protein concentration in that volume, which in turn is measured by staining with Amido Black), then with a simple iterative calculation on the basis of suitable calibration curves, it is easily possible to determine a protein concentration in mg/ml even in an unknown volume between 0.5 and 5 microliters.

[Direct immunological identification of proteins following electrophoretic separation in polyacrylamide micro-gradient gels and the effect of detergents on immunoprecipitation (author's transl)]. / Direkte immunologische Identifizierung von Proteinen nach elektrophoretischer Fraktionierung in Polyacrylamid-Mikrogradientengelen und Eingluss von Detergentien auf die Immunpräzipitation.

Methods for direct immunological identification of single protein components after fractionation of a protein mixture in microgels are described. Protein mixtures were separated with high resolution in polyacrylamide microgradient gels and transferred after electrophoresis into agarose layers containing suitable antisera. Monospecific as well as polyvalent antisera were used. The formation of immunoprecipitates could be observed within approx. 1 h. Immunoprecipitates are also formed in the presence of sodium dodecylsulfate or other detergents, thus allowing immunoreactions to be performed with water-insoluble proteins. Staining of the proteins in the gels did not completely inhibit the immunoreaction, while dansylation of proteins had no effect. The influence of different detergents e.g. sodium dodecylsulfate, Triton X-100, Brij 99, np-40 and urea, as well as different reducing agents e.g. mercaptoethanol, dithiothreitol, thioglycolic acid, on two-dimensional microdiffusion was also studied. When suitable concentrations of these compounds were used, the formation of immunocomplexes was observed within approx. 1 h. This technique can also be applied to immunoreactions with water-insoluble proteins dissolved in detergents.