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Disc electrophoresis has provided a suitable method for separating macromolecules ( Davis, 1964 ; Ornstein, 1964 ). Numerous authors (2,5–10, 12) have presented scaled-down versions which were not directly comparable to the standard separations. In the present study, we attempted to modify the scaled-down procedure by a minimum number of departures from the standard procedure to produce separations which would be directly comparable to the standard separations. We have used the character of the separation of the slow haptoglobins of human serum relative to albumin and transferrin to gauge the performance of our scaled-down procedure. This paper presents a microtechnique which permits the analysis and resolution of serum proteins typical of the standard procedure but with an approximately 20 times reduction in the size of the required protein samples.

A simple microtechnique for the endotoxin assay with Limulus amebocyte lysate is described. Mixtures of 1 μl of reagent with 1 μl of test sample are incubated in 5-μl capillaries which are subsequently dipped into a dye solution. In the absence of a firm gel the dye enters the capillaries whereas a firm gel, formed in the presence of endotoxin, prevents the stain from entering the capillaries. The technique offers a great saving of reagent; unequivocal distinction between positive and negative results, a good reproducibility, and a sensitivity equal to or higher than those in previously reported techniques could be demonstrated.

An improved microtechnique designed for dialysis of solutions with volumes ranging from less than 10 microliter up to approximately 600 microliter is described. Samples, dispensed in Microfuge tubes, are dialyzed in situ across dialysis membrane secured over the tube opening by a perforated Microfuge tube cap. The retentate is efficiently recovered by centrifugation at 10,000g for 10 s. Fifty percent escape (E50) times of [14C]glycine from 25-microliter solutions of soybean trypsin inhibitor (0.1, 1.0, 4.0 mg/ml) in 0.2 M NaCl were approximately 19 min. The E50 times of 3H2O increased in a linear fashion from 2.7 min for 25-microliter samples to 75 min for 600-microliter samples of H2O (pH 7.0, 4 degrees C). The mean permeability coefficient (P) of the dialysis membrane to 3H2O during microdialysis, calculated as 3.0 X 10(-4) cm/s, was similar to membrane permeability coefficients reported for dialysis by conventional methods. Quantitative recoveries (greater than approximately 90%) of [14C]glycine-labeled type I collagen, [methyl-14C]antithrombin III, and [32P]DNA were achieved after microdialysis.

A micromethod was developed for the qualitative and quantitative determination of the glycosaminoglycans of vascular tissues. This procedure requires only 1 mg of dry, defatted tissue and is characterized by its simplicity, high reproducibility, and high resolution of the glycosaminoglycans. Proteolytic digestion with papain and Pronase of the tissue is sufficient for the preparation of the glycosaminoglycans. No dialysis or purification is required prior to the direct determination of these macromolecules. The separation of the glycosaminoglycans is achieved by two-dimensional electrophoresis on cellulose acetate plates. Subsequently, the cellulose acetate plates are stained with alcian blue. For quantification, the dye bound to each GAG (1–10 μg) is determined spectrophotometrically. Thus, the GAG distribution and total GAG content of an aortic or arterial specimen is obtained in a single analysis.

Isoelectric focusing in a density gradient can be performed on chromatographic columns of 10–15-ml volume. A polyacrylamide salt bridge was used to make electrical contact between the density gradient tube and the electrode compartment. An appropriate gradient mixer can easily be made from two 10-ml syringes. The small column permits a resolution of isoenzymes which is comparable to the separation with a commercial 110-ml column. With the described apparatus one uses ten times less Ampholine and biological sample. The time of isoelectric focusing is reduced three to four times.

A microtechnique has been developed for the measurement of alkaline phosphatase in minute amounts of renal tissue. This microtechnique utilizes the known fluorescent property of 4-methylumbelliferyl phosphate following enzymatic hydrolysis. The reaction is sensitive and reproducible and is inhibited by l -bromotetramisole, a specific alkaline phosphatase inhibitor. The microdetermination of alkaline phosphatase activity in the various segments of the mouse nephron allowed the localization of the enzyme in the glomeruli, and in the proximal convoluted tubule where the activity progressively decreases from the capsule of Bowman to the more distal segments. The enzyme was absent from the pars recta or S3 and from the rest of the nephron. This technique is applicable to very small amounts (0.1 μg of protein) of any tissue containing alkaline phosphatase.

A simple microtechnique has been developed for the accurate determination of DNA in less than 1 × 10 6 cultured human fibroblasts or lymphoblasts. This technique involves rapid separation of DNA from the cell extract supernatant and from the extraction buffer in order to avoid interference with the colorimetric diphenylamine assay. The method described does not involve complicated manipulation of samples and does not require special instrumentation. In addition, multiple samples can be conveniently prepared to be assayed at a later time and various other biochemical determinations such as enzyme assays can be done on soluble extracts of the same cell samples.

No simple documented method of sample preparation is available for the analysis of calcium and magnesium in biological samples despite increasing awareness of the biological roles of these cations. The technique described here is rapid, avoids the use of dangerous reagents or costly equipment, and allows accurate determination of protein, calcium, and magnesium content of the speciment after sample preparation. Tissue is solubilized in 1 n NaOH after which one aliquot is used for protein analysis by the method of Lowry et al. (1951) (J. Biol. Chem.193, 265–275), and another is used for determination of cations by atomic absorption spectroscopy. The method was used to analyze biological samples including adipocyte subcellular fractions, bovine liver, and orchard leaves. Results correlated well with those using reference wet ashing and low-temperature ashing techniques with correlation coefficients (r2) of 0.95 for calcium and 0.997 for magnesium. Intraassay coefficients of variation were 4–4.2%. The base-digestion technique is a simple, rapid, and precise method which avoids most of the limitations of currently available sample preparation techniques.