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71.
Quantitative analysis of a ubiquitin‐dependent substrate using capillary electrophoresis with dual laser‐induced fluorescence
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Hyunjung Lee Eunmi Ban Eunice EunKyeong Kim Young Sook Yoo Daekee Lee Eun Joo Song 《Electrophoresis》2014,35(20):2978-2985
Protein degradation by the ubiquitin‐proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS‐dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)‐fusion of the ubiquitin substrate ribophorin 1 (GFP‐RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP‐RPN1 and RFP in cell lysates were performed in an untreated capillary (75 μm × 50 cm) and 100 mM Tris‐CHES buffer (pH 9.0) containing 10 mM SDS. GFP‐RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP‐RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP‐RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS‐dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays. 相似文献
72.
Peroxidase immobilized on Amberlite IRA-743 resin for on-line spectrophotometric detection of hydrogen peroxide in rainwater 总被引:1,自引:0,他引:1
The importance of atmospheric hydrogen peroxide (H2O2) in the oxidation of SO2 and other compounds has been well established. A spectrophotometric method for the determination of hydrogen peroxide in rainwater is proposed. This method is based on selective oxidation of hydrogen peroxide using an on-line tubular reactor containing peroxidase immobilized on Amberlite IRA-743 resin. The hydrogen peroxide in the presence of phenol, 4-aminoantipyrine and peroxidase, produces a red compound (λ = 505 nm). Beer's law is obeyed in a concentration range of 1–100 μmol l−1 hydrogen peroxide with an excellent correlation coefficient (r = 0.9991), at pH 7.0, with a relative standard deviation (R.S.D.) <2%. The detection limit of the method is 0.7 μmol l−1 (4.8 ng of H2O2 in a 200 μl sample). Measurements of hydrogen peroxide in rain samples were carried out over the period from November 2003 to January 2005, in the central area of the Juiz de Fora city, Brazil. The concentration of H2O2 varied from values lower than the detection limit to 92.5 μmol l−1. The effects of the presence of nonseasalt (NSS) SO42−, NO3− and H+ in the concentration of hydrogen peroxide in the rainwater had been evaluated. The average concentrations of H2O2, NO3−, NSS SO42− and SO42− are 23.4, 18.9, 7.9 and 10.3 μmol l−1, respectively. The pH values for 82% of the collected samples are greater than 5.0. The spectrophotometeric method developed in this work that uses enzyme immobilized on the resin ion-exchange compared with the amperometric method did not present any significant difference in the results. 相似文献