We examined the effect of diethylstilbestrol(DES) administration on newborn male rats by rapid micro two-dimensional polyacrylamide gel electrophoresis (RM2D-PAGE) under non-denaturing/denaturing conditions and a 10-17% second-dimensional gel. We began by visually analyzing the changes at each week over time (weeks 1-9) in plasma protein spots from the same individual DES-administered male rats. We found the tendency for mercaptoalbumin (reduced form) spots to increase in week 1 in DES-administered rats. We also analyzed the quantitative alterations in α_2u-globulin (AUG), a biomarker of endocrine disruptors, and found a two-week delay in expression in DES-administered rats in comparison to those not given DES (control rats). We then investigated the reproducibility of these results by using samples taken from multiple male rats in weeks 5, 7 and 9 to perform electrophoretic analysis three times for each individual, and observed the changes in AUG. AUG production was not seen in week 5 in the control rats, but was present at weeks 7 and 9. In DES-administered rats, expression was not observed at week 5 or 7, but was present at week 9. In addition, we identified the spots of AUG by Western blotting after electrophoresis.
 We consequently confirmed that AUG production delayed by two weeks in DES-administered rats, in comparison with control rats, which suggests that the effect of either a decline in AUG synthesis function or its depletion results in stunting of the reproductive organs, which was confirmed through autopsy.

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A miniaturized two-dimensional gel electrophoresis (2-DE) system was constructed with a multifilament-supporting (MFS) gel as a first-dimensional gel for isoelectric focusing (IEF). The MFS gel was 1 mm in diameter, had a multifilament yarn of acrylic as a gel-support, and could provide low-concentration (2.5%T) polyacrylamide with sufficient mechanical strength, resulting in fast IEF, and clear 2-D spots due to the relatively large pore and improved transfer efficiency from the first-dimensional gel to the second-dimensional one of especially high-molecular-weight (HMW) proteins. In this study, by using the MFS gel of 4 cm in length as a miniaturized IEF gel, three colored proteins were separated in 8 min, which was about twice faster than using the MFS gel of standard length (7 cm). The second-dimensional gel was also miniaturized to be 55 mm wide, 40 mm long and 0.75 mm thick. The low-concentration MFS gel after IEF was easily transferred onto the top of the miniaturized second-dimensional gel with a tweezers. By using the miniaturized 2-DE system, the HMW size marker was separated in 30 min by IEF and in 25 min by native-polyacrylamide gel electrophoresis (Native-PAGE), which was at least twice faster than by using the standard size 2-DE system using MFS gel. This miniaturized 2-DE system could be expected as a useful separation method for fast protein diagnosis and screening.

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A vacuum-driven antibody-reaction system enables high-throughput western blotting, where the antibodies pass through protein-transferred membrane and antibody-antigen reaction is completed within 30 min. With the vacuum-based device, two protein detection methods compatible with by laser scanning were compared; one with enhanced chemiluminescence reaction and the other using fluorescence-labeled antibody. Considering sensitivity, quantitative capability, reproducibility and labor intensiveness, we concluded that fluorescence-based detection is more suitable in combination with the vacuum-driven antibody-reaction system and a laser scanner.

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