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(12) and 3.2 times greater than the very sensitive method Comparison of Three Chemiluminescent of Saheki et al. (13), in which the phosphomolybdate com-Horseradish Peroxidase Substrates for plex is reduced by ascorbic acid. At this point, it must be Immunoblotting emphasized that the ascorbic acid procedure has been proposed as an alternative of the radioisotopic phosphorylase assay (13). In addition, in the latter two methods David L. Mattson and Thomas G. Bellehumeur the enzyme was assayed in the presence of saturating Department of Physiology, Medical College of Wisconsin, (20 mM) glucose-1-P concentrations. Thus, our malachite 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226 green procedure is by far the least cost consuming.
We finally applied the malachite green assay for the Received May 14, 1996 measurement of phosphorylase activity in human serum. Samples of blood serum from healthy individuals were Chemiluminescent substrates allow nonradioacmixed with various amounts of skeletal muscle of glycotive, sensitive detection of targeted protein in immugen phosphorylase b and extensively dialyzed against the noblotting protocols (1, 2). The present studies were assay buffer or passed through a small column of alumiperformed to compare three commercially available num oxide (500 mg/ml serum) to remove endogenous horseradish peroxidase (HRP) 1 substrates: ECL phosphate. This step had no effect on added phosphory-(Amersham, Arlington Heights, IL), SuperSignal lase activity. By keeping serum dilutions to a minimum (Pierce, Rockford, IL), and SuperSignal ULTRA (Γ1.5-fold) we were able to detect down to 15 ng/ml of (Pierce). Each substrate consists of luminol and H 2 O 2 glycogen phosphorylase in serum.
with undisclosed chemical enhancers and produces In conclusion, the present procedure may be suitable a luminescent reaction with HRP (manufacturers' for the repeated measurement of serum glycogen phosinstructions). phorylase in the realm of clinical diagnosis.
The three HRP substrates were compared by blotting duplicate amounts of protein onto nitrocellulose membranes and incubating the membranes with primary REFERENCES and secondary antibody. The membranes were then divided and the individual sections were simultane-