SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2020
·
IF 3.232
Na Li,Xing Hu,Junhui Pan,Ying Zhang,Deming Gong,Guowen Zhang
4–Octylphenol is an endocrine disruptor , belonging to environmental estrogens. It can be enriched in the human body through the food chain and may harm human health. Herein, we used a variety of spectroscopic techniques, molecular docking, and gel electrophoresis to study the interaction of 4–octylphenol and ctDNA . It was found that the mechanism of ctDNA quenching the endogenous fluorescence of 4–octylphenol was static quenching, and formed a complex. The negative enthalpy change (Δ H °), entropy change (Δ S °) and Gibbs free energy (Δ G °) have shown that 4–octylphenol and ctDNA spontaneously bind together under the action of hydrogen bonds and van der Waal's force . Viscosity, melting temperature and iodide quenching experiments showed that 4–octylphenol acted on the groove of ctDNA. Insignificant change in circular dichromism spectra further confirmed this binding mode. The binding sites and groups for 4–octylphenol and ctDNA interaction were identified by molecular docking. Gel electrophoresis found that 4–octylphenol at high concentrations caused DNA cleavage. Above findings may lay a theoretical foundation for understanding the toxicity mechanism of 4–octylphenol.
研究领域:
生物化学
·
毒理学
·
环境科学
✓ 已验证
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
·
2014
·
IF 2.694
Dong-Sheng Lian,Shu-Jin Zhao
To develop a highly sensitive method for analyzing nucleic acids using capillary gel electrophoresis with ultraviolet detection (CGE-UV), we combined matrix field-amplified with head-column field-amplified stacking injection (C-FASI) to employ the advantages of two methods. Without diminishing the resolution, a limit of detection of 0.13 ng/ml (signal/noise = 3) in a 300,000-fold diluted sample was obtained, the sensitivity is 102,308 times higher than that achieved with normal pressure injection, 3077 times that with normal electrokinetic injection, 154 times that with pressure field-amplified sample stacking injection, and 31 times that with matrix field-amplified stacking injection. After establishing the method, we tested the detection of a φX174-Hae III digest DNA product without purification and with a high ionic strength. At the lowest dilution of 5000-fold, sample at a concentration of 10 ng/ml was enriched and detected. The relative standard deviations for migration time and peak area ( n = 3) were 0.03–1.15 and 0.72–6.42, respectively. To further validate C-FASI was applicable for real sample, a 400 bp PCR product without purification was directly detected with a limit of detection at the concentration of 6000-fold dilution (signal/noise = 3), The relative standard deviations for migration time and peak area ( n = 6) were 0.44 and 4.8, respectively. These results indicated that C-FASI had good qualitative and quantitative detection abilities and CGE-UV based on C-FASI is easy to perform, practical, highly-sensitive and robust for nucleic acid detection, which makes it a highly valuable tool for genetic diagnostics based on nucleic acid analysis.
研究领域:
生物化学
·
分子生物学
·
分析化学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Zixian Yu,Yiren Cao,Lei Wang,Youli Tian,Rui Zou,Keer Chen,Weiwen Liu,Qiang Zhang,Qingjiang Wang,Baohong Zhang,Chengxi Cao
Monoclonal antibodies (mAbs) are widely used in tumor and autoimmune disease therapy and clinical diagnosis, but they suffer from self-aggregation, particularly dimer formation, impacting their stability, efficacy, and potentially causing severe allergies. Traditional methods for detecting mAb dimers, such as TEM, AUC, DLS, SEC, and CE, are limited by low throughput, high costs, and qualitative data, making them unsuitable for large-scale sample assays in biopharmaceuticals industry as well as antibody research. To address these issues, we developed a facile method of protein cross-linking gel electrophoresis with online intrinsic fluorescence imaging (PC-GE-IFI) for self-aggregation and stability assays of mAbs. This method enables the real-time quantification of monoclonal antibody (mAb) monomers and dimers with exceptional sensitivity, characterized by low detection limits (monomer: 0.9 nM; dimer: 0.45 nM) and a broad dynamic range (monomer: 2.50–2500 nM; dimer: 1.25–1250 nM). Furthermore, the sample wells can be used as windows for the assay of precipitates formed by mAb aggregation. The method supports accurate assessment of mAb dimerization and monomer purity under various stress conditions, including thermal stress, mechanical agitation, and freeze–thaw cycles. Moreover, the method allows concurrent analysis of dimers and precipitates across multiple samples at different concentrations, while nonlinear fitting provides the dissociation constant (Kd) for monomer–dimer interactions, a critical parameter that aids in assessing aggregation propensity, and informs the design and development of mAb products The PC-GE-IFI method has great potential for development, quality control, and safety assessment of mAbs, bispecific antibodies, ADCs, and protein drugs.
研究领域:
生物技术
·
药物开发
·
分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2025
·
IF 9.8
Yiren Cao,Youli Tian,Ke-Er Chen,Yixin Ma,Qiang Zhang,Weiwen Liu,Zhishen Mu,Shengpeng Su,Chengxi Cao
Herein, we report a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method featuring dual imaging and signal-fusion deep learning for specific identification and analysis of glycomacropeptide (GMP) in milk sample. Conventional SDS-PAGE methods lack specificity because of the signle staining of protein bands, and the overlap between GMP and β-lactoglobulin (βLg). Our dual imaging method generated a pair of complementary detection signals by recruiting intrinsic fluorescence imaging (IFI) and silver staining. Comparing the IFI image with the staining image highlighted the presence of GMP and differentiated it from βLg. Additionally, we trained a signal-fusion deep learning model to improve the quantitative performance of our method. The model fused the features extracted from the paired detection signals (IFI and staining) and accurately classified them into different mixing ratios (proportion of GMP-containing whey in the sample), indicating the potential for quantitative analysis on the mixing ratios of GMP added into whey sample. The developed method has the merits of specificity, sensitivity and simplilcity, and has potential to analysis of protein/peptides with unique IFI properties in food safety, basic research and biopharming etc.
研究领域:
生物化学
·
食品科学
·
人工智能
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产品关联
DOI
PubMed
高影响因子
Materials Express
·
2023
·Vol. 13
·期 12
·2057-2063
·
IF 0.7
Ye Jing,Hu Tianxiang,Ding Hui,Dong Xiawei
In clinical practice, many diseases can lead to changes in serum albumin concentration (HSA) in patients. Accurate detection of HSA concentration is of great significance for disease diagnosis. Based on this, this study designed and synthesized 9-(4-amino-aniline)-acridine (AAA) as
a fluorescent probe. By laser induction and capillary gel electrophoresis (CGE), a new rapid and highly specific HSA detection method based on fluorescence-CGE was established. Various experimental control factors were investigated, and the optimal experimental conditions were determined as
follows: the running buffer was H3PO4–KH2PO4 (pH = 2.45, 15.0 mmol L−1), the separation voltage was 30 kV, and the experimental temperature was 25 °C. The sample solution injected 10 s with hydrodynamic mode (3.43×103
Pa), HSA could be directly determined by fluorescence-CGE method. The linear range was 0.10–1.0 μg L−1, the detection limit was 0.012 μg L−1, the relative standard deviation (RSD) was less than 0.30%. This method can be used for the
determination of real HSA samples. In addition, in the presence of various biological macromolecules, small molecules, ions and ethanol, the accurate detection of HSA by fluorescence-CGE method will not be affected, suggesting that this method has a high specificity for HSA. The rapid and
highly specific fluorescence-CGE method of HSA constructed in this study provides a new way to detect HSA, which is of great significance for the diagnosis of clinical diseases.
研究领域:
生物化学
·
医学诊断
·
分析化学
✓ 已验证
DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2024
·
IF 5.7
Zixian Yu,Yiren Cao,Youli Tian,Weicheng Ji,Ke-Er Chen,Zihao Wang,Jicun Ren,Hua Xiao,Lu Zhang,Weiwen Liu,Liuyin Fan,Qiang Zhang,Chengxi Cao
The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution . Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03–10 μg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.
研究领域:
生物化学
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2013
·Vol. 405
·期 26
·8587-8595
·
IF 3.659
Tang Yun-Yun,Wang Hou-Yu,Chen Lu,Li Si,Guo Chen-Gang,Fan Hui-Zhi,Cao Cheng-Xi,Fan Liu-Yin
We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris–Gly), sample buffer (pH 6.78 Tris–Gly), and separation buffer (pH 8.52 Tris–Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below p I values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA 1c , HbA 0 , and HbA 2 ) in MRB-PAGE, in contrast to only one protein zone (HbA 0 ) in ITP-PAGE for large-volume loading (≥15 μl), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory.
Diagram of MRB-induced stacking in a slab PAGE. ( A ) arrangement of separation buffer (pH 8.01–9.55 Tris–Gly), sample buffer (pH 6.37–7.22 Tris–Gly), and electrode buffer (pH 8.21–9.05 Tris–Gly); ( B ) initial MRB formed between electrode and sample buffers for stacking of low-content Hbs in sample buffer under electric field; ( C ) MRB moving toward the anode and partly stacking of Hbs within the MRB; ( D ) quasi-complete stacking of Hbs via MRB closing to the separating gel; ( E ) separation of Hbs in a zone electrophoresis mode
研究领域:
生物化学
·
医学检测
·
蛋白质分析
✓ 已验证
DOI
高影响因子
Scientific Reports
·
2021
·Vol. 11
·期 1
·1-9
·
IF 4.38
Dong Wenjing,Song Erqun,Song Yang
The acute liver injury (ALI) and hepatic fibrosis caused by the co-treatment of lipopolysaccharide (LPS)/ d -galactosamine (D-GalN) have been extensively studied. However, whether LPS/D-GalN are genotoxic has been left unknown. In this study, male mice were divided into eight groups with eight animals in each group. For acute challenge of LPS/D-GalN, the mice in each group received a combination of LPS/D-GalN via intraperitoneal injection at the dose of 25 μg/kg/250 mg/kg, 25 μg/kg/500 mg/kg, or 50 μg/kg/500 mg/kg body weight. An additional group for chronic administration of test compounds was conducted by i.p. injection of LPS/D-GalN (10 μg/kg/100 mg/kg) every other day for 8 weeks. Saline solution (0.9%) and cyclophosphamide (CTX) (50 mg/kg body weight) given by i.p. injection was used as the negative and positive control, respectively. The results of single cell gel electrophoresis (SCGE) assay indicated that acute exposure of the mice to LPS/D-GalN caused severe DNA damage in hepatic cells, but not in the brain, sperm or bone marrow cells, which evidenced the genotoxicity of LPS/D-GalN administrated in combination. Interestingly, the chronic administration of LPS/D-GalN triggered significant genotoxic effects not only in hepatic but also in brain cells, with negative results in sperm and bone marrow cells. Histopathological examination in the liver and brain tissues revealed changes consistent with the SCGE results. The present study indicates genotoxic potential of LPS/D-GalN co-administered in mice, which may serve as an in vivo experimental model for relevant genotoxic study.
研究领域:
毒理学
·
分子生物学
·
医学
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产品关联
DOI
PubMed
高影响因子
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY
·
2021
·
IF 1.134
Xing WEI,Jing-Jing ZHOU,Jin-Hui LIU,Ming-Li CHEN,Jian-Hua WANG
Chromium plays an irreplaceable role in the life system. The exploration of chromium is beneficial to reveal its physiological or pathological processes. In this study, a novel method was established for determination of chromium in gels by matrix-matched external standard method-laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The relative standard deviation of chromium signal intensity during ablation standard were less than 16% ( n = 300), the correlation coefficient ( R 2 ) of LA-ICP-MS quantitative calibration curve was 0.9805, and the detection limit was 2.22 µg/g. The MCF-7 cells were used as an in vitro cell model, and two kinds of polyacrylamide gel electrophoresis (PAGE) method were used to separate the protein extract after chromium incubation. And then, the electrophoresis bands obtained by two electrophoresis methods were ablated respectively by LA-ICP-MS with line scan mode, and the weak chromium-containing protein bands were obtained in electrophoresis band. Finally, the LA-ICP-MS was used to measure and image the chromium in individual cell, and it was found that the cell element distribution map overlapped well with the cell contour map under the laser microscope.
研究领域:
分析化学
·
生物医学
✓ 已验证
产品关联
DOI
高影响因子
ELECTROPHORESIS
·
2016
·
IF 2.482
Shumin Chen,Meiling Wen,Shujie Bu,Ahui Wang,Ya Jin,Wen Tan
Plasma samples from adult male rats were separated by nondenaturing micro 2DE and a reference gel was selected, on which 136 CBB-stained spots were numbered and subjected to in-gel digestion and quantitative LC-MS/MS. The analysis provided the assignment of 1–25 (average eight) non-redundant proteins in each spot and totally 199 proteins were assigned in the 136 spots. About 40% of the proteins were detected in more than one spot and 15% in more than ten spots. We speculate this complexity arose from multiple causes, including protein heterogeneity, overlapping of protein locations and formation of protein complexes. Consequently, such results could not be appropriately presented as a conventional 2DE map, i.e. a list or a gel pattern with one or a few proteins annotated to each spot. Therefore, the LC-MS/MS quantity data was used to reconstruct the gel distribution of each protein and a library containing 199 native protein maps was established for rat plasma. Since proteins that formed a complex would migrate together during the nondenaturing 2DE and thus show similar gel distributions, correlation analysis was attempted for similarity comparison between the maps. The protein pairs showing high correlation coefficients included some well-known complexes, suggesting the promising application of native protein mapping for interaction analysis. With the importance of rat as the most commonly used laboratory animal in biomedical research, we expect this work would facilitate relevant studies by providing not only a reference library of rat plasma protein maps but a means for functional and interaction analysis.
研究领域:
蛋白质组学
·
生物化学
·
医学研究
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY
·
2022
·
IF 7.129
Huixia Zhang,Zhaomin Wang,Yong Liu,Ping Xie
Microcystin-leucine arginine (MC-LR) is the most toxic and abundant microcystin produced by cyanobacteria. Previous studies have demonstrated that MC-LR can lead to DNA damage by increasing intracellular reactive oxygen species content to induce oxidative stress. However, the direct effect of MC-LR on DNA has not been fully described. In this study, the direct effect of MC-LR on DNA was explored by using spectral analysis and molecular biology technology. First, the fluorescent probe Bptp-R2 was developed to monitor different types of DNA and explore the direct interaction between DNA and MC-LR. The significant differences in the fluorescence of probe–plasmid DNA and probe–ds DNA at various MC-LR concentrations (0, 5, 10, 20, and 30 μmol/L) and MC-LR exposure times (0, 6, 12, and 24 h) showed that the direct interaction between DNA and MC-LR was significant (P ≤ 0.01). Gel electrophoresis demonstrated that the direct interaction between DNA and MC-LR cannot cause DNA strand breaks or change DNA configuration. Then, PCR experiments revealed that the direct interaction between DNA and MC-LR cannot affect DNA replication in a PCR system (P ≤ 0.01). This study discovered that the effects of MC-LR on DNA originate mainly from the secondary effects of MC-LR rather than from the direct interaction between DNA and MC-LR.
研究领域:
毒理学
·
分子生物学
·
环境科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Nature Protocols
·
2017
·Vol. 12
·期 10
·2147-2168
·
IF 10.032
Yang Yi,Song Haiping,He Dan,Zhang Shuai,Dai Shizhong,Xie Xiao,Lin Shixian,Hao Ziyang,Zheng Huangtao,Chen Peng R
Although protein–protein interactions (PPIs) have crucial roles in virtually all cellular processes, the identification of more transient interactions in their biological context remains challenging. Conventional photo-cross-linking strategies can be used to identify transient interactions, but these approaches often suffer from high background due to the cross-linked bait proteins. To solve the problem, we have developed membrane-permeable releasable photo-cross-linkers that allow for prey–bait separation after protein complex isolation and can be installed in proteins of interest (POIs) as unnatural amino acids. Here we describe the procedures for using two releasable photo-cross-linkers, DiZSeK and DiZHSeC, in both living Escherichia coli and mammalian cells. A cleavage after protein photo-cross-linking (CAPP ) strategy based on the photo-cross-linker DiZSeK is described, in which the prey protein pool is released from a POI after affinity purification. Prey proteins are analyzed using mass spectrometry or 2D gel electrophoresis for global comparison of interactomes from different experimental conditions. An in situ cleavage and mass spectrometry (MS)-label transfer after protein photo-cross-linking (IMAPP) strategy based on the photo-cross-linker DiZHSeC is also described. This strategy can be used for the identification of cross-linking sites to allow detailed characterization of PPI interfaces. The procedures for photo-cross-linker incorporation, photo-cross-linking of interaction partners and affinity purification of cross-linked complexes are similar for the two photo-cross-linkers. The final section of the protocol describes prey–bait separation (for CAPP) and MS-label transfer and identification (for IMAPP). After plasmid construction, the CAPP and IMAPP strategies can be completed within 6 and 7 d, respectively.
研究领域:
生物化学
·
分子生物学
·
细胞生物学
·
蛋白质组学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）