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Two fluorophore-nitrilotriacetic acid conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro-Q Sapphire 365 oligohistidine gel stain exhibits bright-blue fluorescence (emission maximum = 450 nm) when illuminated with UV-A or UV-B light from a standard ultraviolet transilluminator. Pro-Q Sapphire 488 oligohistidine gel stain exhibits bright-green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser-based gel scanner equipped with a 470 nm second-harmonic generation (SHG) or 488 nm argon-ion laser source. Typically, 25-65 ng of oligohistidine-tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro-Q Sapphire dyes, gels may be post-stained with the red-fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern. 相似文献
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To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies. 相似文献
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Ju Wang Wei Yao Di Fan Zhenjun Qiu Jieqiong Song Kai Pan Taijun Hang 《Biomedical chromatography : BMC》2019,33(10)
HR011303 is a novel and highly selective urate transporter 1 (URAT1) inhibitor. In this study, a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantification of HR011303 in beagle dog plasma. Plasma samples were pretreated with protein‐precipitation extraction by acetonitrile and added with a trifluoromethyl substituted analog of HR011303 as internal standard. The chromatographic separation was performed on a Shiseido C18 column (100 × 4.6 mm, i.d., 5 μm) by mobile phases consisting of 5 mm ammonium–formic acid (100:0.1) and acetonitrile–formic acid (100:0.1) solutions in gradient elution. The MS detection was conducted in electrospray positive ionization with multiple reactions monitoring at m/z 338 → 240 for HR011303 and m/z 328 → 230 for the internal standard using 25 eV argon gas collision induced dissociation. The established LC–MS/MS method showed good selectivity, sensitivity, precision and accuracy. The plasma pharmacokinetics of HR011303 in beagle dogs following both oral and intravenous administration were then successfully evaluated using this LC–MS/MS method. 相似文献
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Xiaoyu Dong Liang Cui Yunjing Shi Shilie Pan Zhongxiang Zhou Zhihua Yang Bingbing Zhang Xiangzhan Jiang Yun Yang Zhaohui Chen Zhenjun Huang 《无机化学与普通化学杂志》2013,639(6):988-993
The single crystals of Ba2Cd(B3O6)2 were grown by the spontaneous crystallization method for the first time. They crystallize in the centrosymmetric trigonal space group R$\bar{3}$ with a = 7.143(3) Å, c = 17.405(16) Å, and Z = 3. The structure is characterized by isolated B3O6 units, and the Ba2+ and Cd2+ cations connect with B3O6 rings to form three dimensional structure. The TG/DSC and XRD results reveal that Ba2Cd(B3O6)2 melts congruently. First‐principles electronic structure calculation performed with the density functional theory (DFT) method shows that the calculated bandgaps are 4.66 eV, which is in good agreement with the UV/Vis/NIR experimental value 4.59 eV. The calculation shows that the Ba2Cd(B3O6)2 crystal has a large birefringence (Δn = 0.0875–0.0569 from 270 nm to 2600 nm), which demonstrates that Ba2Cd(B3O6)2 is a potential birefringence crystal. 相似文献
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Xiyu Liao Zhenjun Zhu Shujian Wu Mengfei Chen Rui Huang Juan Wang Qingping Wu Yu Ding 《Molecules (Basel, Switzerland)》2020,25(22)
Pleurotus geesteranus is a promising source of bioactive compounds. However, knowledge of the antioxidant behaviors of P. geesteranus protein hydrolysates (PGPHs) is limited. In this study, PGPHs were prepared with papain, alcalase, flavourzyme, pepsin, and pancreatin, respectively. The antioxidant properties and cytoprotective effects against oxidative stress of PGPHs were investigated using different chemical assays and H2O2 damaged PC12 cells, respectively. The results showed that PGPHs exhibited superior antioxidant activity. Especially, hydrolysate generated by alcalase displayed the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (91.62%), 2,2-azino-bis (3-ethylbenzothia zoline-6-sulfonic acid) (ABTS) radical scavenging activity (90.53%), ferric reducing antioxidant power, and metal ion-chelating activity (82.16%). Analysis of amino acid composition revealed that this hydrolysate was rich in hydrophobic, negatively charged, and aromatic amino acids, contributing to its superior antioxidant properties. Additionally, alcalase hydrolysate showed cytoprotective effects on H2O2-induced oxidative stress in PC12 cells via diminishing intracellular reactive oxygen species (ROS) accumulation by stimulating antioxidant enzyme activities. Taken together, alcalase hydrolysate of P. geesteranus protein can be used as beneficial ingredients with antioxidant properties and protective effects against ROS-mediated oxidative stress. 相似文献
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We synthesized two new compounds: Sodium 2-(4′-dimethyl-aminocinnamicacyl)-3,3-(1′,3′- ethyl- enedithio) acrylate (STAA-1) and Sodium 2-(4′-dimethyl-aminocinnamicacyl)-3, 3-(1′,3′-propylenedithio) acrylate (STAA-2). The maximum absorption of these compounds ranges from 460 to 520 nm with different molecular structures in different solvents. Meanwhile, the emission peak of these compounds arranges from yellow (510 nm) to red (605 nm). The emission spectra show red shift according to the strength of the hydrogen bonding property of the solvent. But the absorption spectra do not show clearly relationship with the strength of the hydrogen bonding property of the solvent. The Stoke shift of the compounds ranges from 42 to 102 nm. It changes in the following order, EtOH>H2O>DMF, and STAA-1>STAA-2 in the same solvent. The fluorescent quantum yield of STAA-1 was measured to be 7.12% with quinine sulphate as the standard compound in ethanol. Furthermore, the relationship of the fluorescence of STAA-1 with pH (ranges form 4 to 14) in water (c=∼10−4) was studied to make sure that these compounds could be used as proton sensors. 相似文献
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