Establishment and Optimization of Two-dimensional Electrophoresis System for Spleen Proteome of Sillago sihama Forsskal

Taking Sillago sihama Forssk?l as the research object,through the optimization of the extraction method,hydration and isoelectric focusing,a two-dimensional electrophoresis system was established for spleen proteome of S. sihama Forssk?l. The results showed that the twpstep hydration and isoelectric focusing method is better than the hydration and isoelectric focusing integrated method. The protein spots of the spleen of S. sihama Forssk?l were detected by two-dimensional electrophoresis mainly at pH 4. 0-7. 0. The establishment of this technical system will lay a foundation for further research on proteomics of S. sihama Forssk?l in the future.

Study on the Optimization of Two-dimensional Electrophoresis Technology System for Rapeseed Proteome

[Objective] The research aimed to establish the two-dimensional electrophoresis（2-DE）technology which was suitable for the rapeseed proteome research.[Method] Xiangyou 17 was as the material.The sample preparation method,gel concentration and loading amount,etc.in 2-DE technology were optimized.[Result] The best extraction method of total protein of rapeseed was TCA-acetone method,and the protein spots on 2-DE map were the most.When IPG strip（pH 3-10）and 12% gel were used,and the loading amount was 250 μg,the two-dimensional electrophoresis map with the clear background,good repeatability and high protein spot resolution was obtained.[Conclusion] The research laid the foundation for carrying out the rapeseed proteomics research.

A Capillary Electrophoresis Detection Scheme for Water-soluble Vitamins Based on Luminol-BrO^- Chemiluminescence System

A novel chemiluminescence detection scheme has been developed for detecting water-soluble vitamins following capillary electrophoresis (CE) separation. This detection was based on the inhibitory effect of vitamins on the CL reaction between luminol and BrO- in basic aqueous solution. Detection of vitamins was accomplished with a boratebased background electrolyte at pH 9.2. The luminol was used as a component of the separation carrier electrolyte.

Highly sensitive trivalent copper chelate-luminol chemiluminescence system for capillary electrophoresis chiral separation and determination of ofloxacin enantiomers in urine samples

A simple,fast and sensitive capillary electrophoresis（CE） strategy combined with chemiluminescence（CL） detection for analysis of ofloxacin（OF） enantiomers was established in the present work.Sulfonated p-cyclodextrin（β-CD） was used as the chiral additive being added into the running buffer of luminol-diperiodatocuprate（Ⅲ）（K[Cu（HIO6）2],DPC） chemiluminescence system.Under the optimum conditions,the proposed method was successfully applied to separation and analysis of OF enantiomers with the detection limits（S/N=3） of 8.0 nM and 7.0 nM for levofloxacin and dextrofloxacin,respectively.The linear ranges were both 0.010-100 μM.The method was utilized for analyzing OF in urine;the results obtained were satisfactory and recoveries were 89.5-110.8%,which demonstrated the reliability of this method.This approach can also be further extended to analyze different commercial OF medicines.

A Compact System of Capacitively Coupled Contactless Conductivity Detection Based on the Square Wave Excitation Signal for Capillary Electrophoresis

This paper reported a compact system of capacitively coupled contactless conductivity detection （C4D） based on the square wave excitation voltage for capillary electrophoresis, and it exhibited excellent sensitivity at the optimal frequency of 198 kHz. The feasibility and sensitivity of this detector was demonstrated by simultaneous detection of thirteen ions including alkali, alkaline earth and heavy metal ions. And the detection limits （S/N 3） were in the range of 0.2-1μmol/L for Mn^2＋, K^＋, Na^＋, Mg^2＋, Ca^2＋, Li^＋, Ba^2＋, and 7-25 μmol/L for Ni^2＋, Cu^2＋, Cd^2＋, Pb^2＋, Co^2＋, Zn^2＋.

Enzyme electrophoresis method in analysis of active components of haemostasis system

The novel modifications of substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis that can be used for the detection of proteases and its activators are reported. The protease/activator samples were separated on a protein substrate-SDS-polyacrylamide gel. To detect plasminogen activators fibrinogen and Glu-plasminogen were incorporated into the SDS-PAG followed by 1 h incubation at 37?C in thrombin solution (1 NIH/ml). After electrophoresis the gel was stained according to the standard protocol. To detect fibrin-unspecific plasminogen activators from snake venom incubation in thrombin solution was substituted for 12 h incubation in 50 mM Tris-HCl (pH 7.4). To detect fibrinogen-degrading enzymes fibrinogen-containing gel was used. Activity of protease/activator was visualized in the gel as clear bands against the dark background. These new techniques offer several advantages including determination of the quantity and activity of t-PA and urokinase, however cannot be recommended for precise quantification of activators;the total procedure is quite quick and simple;method is convenient tool for detection of novel protein-protein interactions in haemostasis system;the sensitivity of the method is ≤0.01 IU per track.

On-Line Multichannel Raman Spectroscopic Detection System For Capillary Zone Electrophoresis

An on-line multichannel Raman spectroscopic detection system for capillary electrophoresis was established by using an Ar+ laser and a cryogenically cooled ICCD. Resonant excitation Raman spectra of methyl red and methyl orange were employed to test the system. The result shows that it could yield on-line electrophoretogram and time series of Raman spectra.

A New Dual-electrode and Multi-channel Electrochemical Detection System for Capillary Electrophoresis

A new type of dual-electrode and multi-channel electrochemical detection technology for capillary electrophoresis is described in this paper. Two detectors(the amperometric detector and the conductometric detector) or two conductometric detectors are connected to the same capillary electrophoresis system. The whole system possesses the advantages of the two electrochemical detectors including sparing time, improving the analytical speed and expanding the sample range. The working electrode and detector cell are handled easily. The system was applied to sample detection with satisfactory results.

Analysis of Resistance-related Proteins in Rice Against Brown Planthopper by Two-dimensional Electrophoresis

A recombinant inbred population (RI) was constructed from a cross between B5, an introgression. line from the wild rice Oryza officinalis Wall. ex Watt, and susceptible cultivar Minghui 63 ( O. sativa L.). The brown planthopper ( BPH) resistances of RI lines were evaluated. Based on bulked segregant analysis (BSA), two protein bulks were made by extracting proteins from equally mixed seedlings of extremely resistant and susceptible plants selected from the RI population, respectively. Two-dimensional electrophoresis was used to detect the changes of polypeptide pattern. Results showed that a protein P40 ( pI 6.3, Mw 40 kD) was significantly reduced or vanished after BPH infestation for 48 h in the susceptible bulk, while it remained uninfluenced in the resistant bulk. In connection with the physiological changes of the resistant and susceptible lines subjected to BPH sucking, we suppose that the protein P40 is related to the interaction responses of lice plants to BPH infestation.

Research on Sex and Tissue Differences in Isozymic Phenotype of Varicorhinus macrolepis through Isozyme Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) and biochemical staining method were used in this study for the analysis on malate dehydrogenase (MDH,E.C. 1.1.1.37) isozymes zymogram in 11 different types of tissues of male and female Varicorhinus macrolepis. It had been found for the first time that the phenotype of malate dehydrogenase (MDH),acid phosphatase (ACP) and superoxide dismutase (SOD) showed difference between male and female V. macrolepis,and there was no difference among different individuals in the same sex. Therefore,the electrophoresis band of malate dehydrogenase,acid phosphatase and superoxide dismutase could be used as an indicator for the identification of gender and tissues of V. macrolepis,which would provide basic data for the developmental genetics,variety improvement and directed breeding of V. macrolepis groups,thus facilitating the development and protection of this valuable fish species.

Interactions Between Thrombin and Natural Proudcts of Coreopsis tinctoria Nuttt. and Cistanche deserticola Ma. in Capillary Zone Electrophoresis

Aim A capillary zone electrophoretic method （CZE） was used to determine the interactions between natural products and thrombin. Methods Samples containing natural products and thrombin at various ratios were incubated at 25 ℃ and then were separated by CZE with Tris-acetate buffer at pH 7.2. Each run could be accomplished within 5 min. Results In CZE, the peak width broadened due to the affinity interaction between natural products and thrombin. Compared with positive and negative control, the natural products （CB-1, CB-2） from Coreopsis tinctoria Nuttt. interacted with thrombin; CB-3 from Coreopsis tinctoria Nuttt. and HC-1, HC-2, HC-3 from Cistanche deserticola Ma. did not bind to thrombin. Both qualification and quantification characterizations of the binding were determined. Conclusion The established method is capable of sensitive and fast determination of natural products and thrombin interactions, it can be employed as an alternative method.

Optimization of SSR-PCR Non-denatured Polyacrylamide Gel Electrophoresis Conditions in Kernelled Apricot

[Objective] The aim was to optimize the SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Method]25 accessions of kernelled apricot and three accessions of edible apricot were selected as experimental materials to screen the repeatable SSR loci with high polymorphism by the use of SSR markers combined with non-denatured polyacrylamide gel electrophoresis.And the effect of different factors on electrophoresis conditions was compared to explore the optimal SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Result]The optimal non-denatured polyacrylamide gel electrophoresis conditions for SSR-PCR were established as follows：polyacrylamide gel concentration 6%,the ratio of acrylamide to bisacrylamide 29∶1,electrophoresis at 1 000 V for 2-3 h,and staining for 15 min within 0.1% AgNO3.[Conclusion]The optimum electrophoresis system has provided some technical foundations to further study the phylogenetic relationship of kernelled apricots by SSR markers.

Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

The Superiority of Like-rocket Immunoelectrophoresis Using in Single Cell Gel Electrophoresis Assay

[Objective] The like-rocket immunoelectrophoresis was used to explore a new feasible electrophoresis method for single cell gel electrophoresis assay （comet assay）.[Method] The like-rocket immunoelectrophoresis was used for single cell gel electrophoresis assay to detect DNA damage at single cell level,then it was compared with traditional electrophoresis method to analyze its advantage and disadvantages.[Result] Under cell DNA undamaged state,the results of two electrophoresis methods were consistent.When cell DNA was damaged,the comet tail divergence of some cells under traditional electrophoresis method were drifted,however,the comet tail image of like-rocket immunoelectrophoresis was concentrated and not shifted.[Conclusion] The like-rocket immunoelectrophoresis had some advantages.

Electrophoresis Analysis on the Protein Expression in Heteromorphic Leaves of Populus euphratica Oliv.

[Objective] This study was to elucidate the cellular and molecular mechanism of the development of heteromorphic leaves of Populus euphratica Oliv. [Method] By employing SDS-PAGE and 2-demensional electrophoresis (2-DE) techniques,proteins in various heteromorphic leaves from the same adult tree of P. euphratica were isolated and separated to the electrophoresis technique suitable for the separation and analysis of proteins in leaves of P. euphratica tree. [Results] There were significant differences in the expressions of proteins in various heteromorphic leaves of P. euphratica tree. SDS-PAGE pattern showed that bands of proteins with molecular weight of 57.2,13.2,30.2,23.9 and 33.3 kDa were remarkably different. 2-D electrophoresis pattern presented that proteins in leaves of P. euphratica tree mainly belong to acidic proteins distributed at pH value of 5.0-6.5 and with molecular weight of 20-40 kDa; totally 73 different protein spots were observed,of which 51 were up expressed and other 22 were down expressed in the serrated ovate leaves. [Conclusion] Based on these results,we speculate that regulated gene expression in leaves of P. euphratica tree results in the generation of different shapes of leaves,in order to adapt to the surroundings better.

Enantioseparation of Several PharmaceuticalRacemates with High PerformanceCapillary electrophoresis

With high performance capillary electrophoresis using -cyclodextrin or its deriveatives as the chiral selectors, five pairs of drug enantiomers were separated, The PH of the back- gmund electrolyte and the chiral selector concentrations were optimized; and the effect of organic modifier on separation of chlorpheniramine enantiomers was also inver

Electrophoresis of poly(dT)_(20) through α-hemolysin nanopore in high concentration potassium chloride solution

Experiments of poly（dT）20 electrophoresis throughα-hemolysin nanopores were performed to unveil the electrophoretic transport mechanism of DNA through nanopores in high concentration potassium chloride solution. It is found that there are two obvious current blockades induced by poly（dT）20 translocation and collision events. Both blockade currents increase linearly with the applied bias voltage. However, the normalized blockade currents are almost kept the same although variable bias voltages are applied. The collision time of poly（dT）20 in the luminal site of the pore remains constant for different voltages. The translocation speed of poly（dT）20through the nanopore decreases with the increase of bias voltage. It is because as the potential increases, the drag force on the homopolymer helps it to crumple into a cluster much easier due to the poor stacking of thymine residues compared with homopolymers consisting of other nucleotides. Molecular dynamics simulations further confirm the experimental results. Increasing the applied bias voltage can slowdown the translocation velocity of the flexible poly（dT）20, which favors increasing the precision of single molecule detection by using nanopores.

PCR-DGGE Analysis of Bacterial Communities Structure in Babylonia areolata Culture Systems of The Subtidal Zone and The Pond Mulched Plastic Film and Sand in Bottom

To know the bacterial communities structure in Babylonia areolata culture systems and to research and optimize the management pattem of Babylonia areola-ta culture systems of the pond mulched plastic film and sand in bottom, the bacte- rial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom were analyzed at molecular level by adopting the denaturing gradient gel electrophoresis （DGGE）. The results indicated that the dominant bacterial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom, which were built on the basis of the seawater in East-island of Zhanjiang, included Proteobac- teda Chloroflexi, Cyanobacteria and Actinobacteria. The dominant bacterial groups in the above pond culture system were Garnmaproteobacteria, Alphaproteobacteria, Deltaprotecbacteda, Epsilonproteobacteda, Anaerolineae, Cyanobacteria and Acti- nobacteda. The dominant bacterial communities in the subtidal zone culture system were Gammaprotecbacteda, Alphaproteobacteria, Deltaproteobacteria, Anaerolineae and Cyanobacteda, and there were less Epsilonproteobacteria and Actinobacteria in the culture system. The higher diversity was detected in the above two culture sys- tems. The results of unweighted pair group method with arithmetic average （UPG- MA） showed that the bacterial communities of the sediment samples S1 and S2 in the above two culture systems were a cluster, the similarity of bacterial communities was 54.5%. The bacterial communities of seawater samples S3 and S4 in the above culture systems were in clusters, and the similarity of the bacterial communi- ties was 84.0%. The results showed that the microorganism ecological level in the Babylonia areolata culture systems of the pond mulched plastic film and sand in bottom could be similar to the sub-tidal zone culture systems through changing the pond seawater and monitoring the microbial population.

A Peptides Migration Model in capillary Zone Electrophoresis

A peptides migration model based on the principle of mechanics is presented in capillary zone electrophoresis (CZE). It is shown that the migration that the (tr) is a function of electric (Q), relative molecular mass (Mr), conformation parameter (Rc) of peptides and electrophoretic condition parameter(A). The conformation parameter is introduced to characterize multifarious shapes owing to the complex conlormation and the various kinds of macromolecules, where Rc≥1/3. The parameters A and Rc can be obtained from experimental data. The times of migration of the nine standard peptides were measured in pH 2.5buffer on different electrophoretic conditions in CZE. The experimental results agreed well with the theoretical prediction.