Background-free,fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes,zincon and ethyl violet

A background-free, fast protein staining method in polyacrylamide gel electrophoresis using an acidic dye, zincon (ZC) and a basic dye, ethyl violet (EV) is described. It is based on the counterion dye staining technique that employs two oppositely charged dyes to form an ion-pair complex in staining solution. The selective binding of free dye molecules to proteins in acidic solution produces bluish violet-colored bands. It is a rapid and end-point staining procedure, involving only fixing and staining steps that are completed in 1-1.5 h. The detection limit of this method is 8-15 ng of protein that is comparable to the sensitivity of the colloidal Coomassie Brilliant Blue G (CBBG) stain. Due to its sensitivity and speed, this stain may be more practical than any other dye-based stains for routine laboratory purposes.     

高效毛细管电泳电导法快速检测复方维生素B片中的VB1、VB12、VB6和VC

采用高效毛细管电泳电导法同时分离、测定了复方维生素B片中的主要成分VB1, VB12,VB6和VC的含量.研究了运行缓冲溶液的酸度和浓度、电泳电压、进样时间等因素对电泳的影响.在优化的实验条件下40 mmol/L Tris -4 mmol/L H3BO3 (pH 8.0) 的缓冲溶液中加入0.30 mmol/L CTAB(溴化十六烷基三甲基铵),分离电压为15 kV,上述4组分在5 min内得到良好的分离.维生素B1,B12,B6和VC的线性范围分别为5.5～1.0 mg/mL; 15～1.5 mg/mL; 1.0～0.40 mg/mL和6.6～0.80 mg/mL; 检测限分别为0.80 μg/mL, 4.0 μg/mL, 0.50 μg/mL, 2.9 μg/mL; 5次测定峰高的相对标准偏差分别为2.2%, 1.6%, 3.9%, 2.8%.5次测定的平均回收率分别为99%, 94%, 100%, 97%.     

Micellar electrokinetic capillary chromatography using polymeric hollow fibers

Summary In this paper, polymeric hollow fibers prepared from pH-stable polypropylene were used as columns for micellar electrokinetic capillary chromatography (MECC). The electroosmotic flow (EOF) for polypropylene hollow fibers was evaluated in the pH range of 5.0–12.0. With untreated polypropylene hollow fibers a stabilized but enhanced EOF was achieved when SDS was used in the buffer, decreasing the separation window for uncharged substances in MECC to impractical levels. Uncharged acrylamide and charged 2-acryloylamido-2-methylpropane sulfonic acid surface modifications were used to lower the strength of the EOF, increase the separation window and prevent local overheating that could melt the column wall.     

Study of the development of thermoresistance in human pancreatic carcinoma cell lines using proteome analysis

In order to find candidate proteins that are potentially associated with the thermoresistant phenotype in combination with drug resistance, we analyzed the differential protein expression in vitro in the human pancreatic cancer cell line EPP85-181-P and classical and atypical multidrug-resistant variants and their thermoresistant counterparts using proteomics. This study identifies sets of proteins that may lead to the development of thermoresistance. These results provide a fundamental basis to elucidate the molecular mechanism of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers.     

Immunoassay of P-glycoprotein on single cell by capillary electrophoresis with laser induced fluorescence detection

The cellular mechanism based on P-glycoprotein (PGP) for its drug pump function has become very important in multidrug resistance (MDR) research. A method has been established to characterize PGP on single K562 cell by coupling capillary electrophoresis with laser induced fluorescence detection. A permeable intact cell after the immunoassay binding with fluorescence labeling antibody was injected into the capillary and directly separated without lysis. It was found that once 5-10 optional cells were detected in batch, the PGP amount on this cell line could be outlined and calculated clearly. The PGP amount on K562 MDR cell line is 3.88 times higher than that on K562 sensitive cell line. These two cell lines with immunoassay binding were also analyzed by injection of multi-cells in order to improve the throughput. A resistance factor so called multidrug resistance multiple (MRM) was introduced to evaluate the MDR difference between cell lines. The MRM values of the cell line K562 measured by single cell analysis are well correlated with those by flow cytometry, which also prove the validity of our method in single cell analysis for the possibility of cancer diagnosis, pharmacokinetics and drug screening in future.     

牙本质中天冬氨酸消旋化程度的测定——手性毛细管气相色谱法与高效液相色谱法的比较

应用手性毛细管气相色谱法（酸性异丙醇、三氟乙酸酐为衍生试剂）、高效液相色谱法（邻苯二甲醛－Ｎ－乙酰－Ｌ－半胱氨酸为手性衍生试剂）同时测定人类第一磨牙牙本质中天冬氨酸的消旋化程度，结果表明，两者均可有效地用于天冬氨酸对映体的分离测定，但气相色谱检测限（０．２ｐｍｏｌ）低于高效液相色谱（１．０ｐｍｏｌ），而变异系数（７％）、总分析时间（１００ｍｉｎ）均高于高效液相色谱（分别对应为４％，３１ｍｉｎ）。     

Comparison between capillary electrophoresis and ion-chromatography for analysis of inorganic anions

Separation and detection of some selected inorganic anions with capillary electrophoresis are shown. The anions are separated in microbore capillaries (25 m* 0.2 m) and detected with a UV-detector. Results are compared with the method of ion exchange chromatography. In consideration of the most important physical and chemical parameters an easy kind of computer simulation for such electropherograms was developed.To get optimal results of separation and UV-detection in capillary electrophoresis some parameters of the device HPE 100, i.e. loading time in the electrokinetic sample injection mode and the running voltage are varied. The behaviour of absorption in the UV region of the chosen anions as well as the influence of pH values in retention behaviour are investigated. There is a simple way to calculate the electrophoretic mobilities from known retention times. Approximate limits of detection for all anions and for each technique are given.     

Indirect UV detection of carbohydrates in capillary zone electrophoresis

Summary A new system for the rapid and sensitive analysis of underivatized carbohydrates has been established using capillary zone electrophoresis with indirect UV detection. At an applied potential of 28 kV, sugars and sugar acids could be separated by the combined effects of electroendosmosis and electrophoresis within 20 minutes in a fused silica capillary of 50 m internal diameter and an effective length of 100 cm using 6mM sorbic acid, pH 12.1, as both carrier electrolytie and chromophore. The alkaline pH ensured ionization of the sugars and, hence, their detection by means of charge displacement. Furthermore, the chosen concentration of sorbic acid allowed the smallest fractional change in the background signal to be measured. While the electrophoretic mobilities of the sugars were found to increase within a pH range of 11.9 to 12.3, those of the sugar acids were not affected. Due to the increasing competition of hydroxide ions in the displacement of the chromophore with rising pH, a significant loss of sensitivity is observed at pH values higher than 12.1 and this pH was found to provide sufficient resolution, optimum sensitivity, and a acceptably short analysis time. Under these conditions, a lower detection limit of 2 pmol was obtained for glucose.     

毛细管电泳法测定白头翁汤中黄连和黄柏共煎生物碱的煎出量

 建立了利用毛细管电泳简便、准确地测定白头翁汤中黄连和黄柏共煎生物碱煎出量的方法。采用自组装毛细管电泳装置,采用75μmi.d.×50cm弹性石英毛细管,以0 05mol/LNa2B4O7 CH3OH(体积比为85∶15)溶液作缓冲液,运行电压为14kV,检测波长为232nm。另外,通过实验优化了提取溶剂中乙醇的含量。实验结果表明:以小檗碱、巴马汀提取量为指标,30%(体积分数)的乙醇水溶液是提取白头翁汤中黄连和黄柏共煎生物碱的最佳溶剂。小檗碱和巴马汀的质量浓度分别在15 0mg/L～65 0mg/L、12 5mg/L～50 0mg/L时与其峰面积有良好的线性关系;小檗碱的平均回收率不低于95%。     

BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).