Quantitative Detection of Inositol Hexakisphosphate (InsP6) in Crop Plants Using Polyacrylamide Gel Electrophoresis (PAGE)

Inositol phosphates are essential for cell development and signaling in all living organisms. Inositol hexakisphosphate (InsP6) is the most abundant phosphoinositol in both plants and animals. While the concentration of inorganic phosphorous (Pi) is often limited in soil, some plants overcome this limitation by creating a phosphate reservoir that serves as a source of Pi during phosphate deficiency. Although this strategy benefits plant development and signaling under adverse environmental conditions, excessive accumulation of Pi in crop plants has raised serious concerns about its toxicity and ill effects on human health. Consumption of crop plants with high InsP6 content or food products made from these crops is found to reduce nutrient intake significantly by way of chelating essential metal cations in human and livestock fed by such plants. Therefore, it is necessary to determine InsP6 contents in crop plants. Several methods have been developed for the screening and detection of InsP6 in plants. These detection methods however, are complex, labor-intensive, and often provide inaccurate results. We have developed a fast, reliable, and cost-effective method for the detection and quantification of InsP6 in plants using polyacrylamide gel electrophoresis (PAGE) with potential applications in industry, quality control labs, and research projects.

Two-dimensional polyacrylamide gel electrophoresis analysis of indomethacin-treated human colon cancer cells

AIM: To establish the two-dimensional gel electrophoresis (2-DE) profiles of indomethacin (IN)-treated human colon cancer cell line HCT116, and to provide a new way to study its anti-tumor molecular mechanism through analyzing a variety of protein maps.METHODS: Two-DE profiles of HCT116 were established in IN-treated and untreated groups. Total proteins were separated by immobilized pH gradient-based 2-DE. The gels were stained by silver, scanned by ImageScanner,and analyzed with Image Master software.RESULTS: Clear background, well-resolved and reproducible 2-DE patterns of HCT116 cells were acquired in IN-treated and untreated group. The average deviation of spot position was 0.896±0.177 mm in IEF direction and 1.106±0.289 mm in SDS-PAGE direction respectively. In IN-treated group,1 169±36 spots were detected and 1 061±32 spots were matched, the average matching rate was 90.6% in three gels. In untreated group, 1 256±50 spots were detected and 1 168±46 spots were matched, the average matching rate was 93.0% in three gels. Forty-five differential protein spots were displayed between IN-treated and untreated groups. Of which, 34 protein spots decreased and 9showed higher expression in IN-treated group, and only two protein spots showed an expression in untreated cells.CONCLUSION: Two-DE profiles of IN-treated and untreated HCT116 cells were established. Apparent 45 different protein spots were detected in IN-treated and untreated HCT116 cells. The analysis on differential protein spots may serve as a new way to study the molecule mechanism of IN-treated colon cancer.

Separation of non-denatured proteins using semi-crosslinked polyacrylamide capillary gel electrophoresis

This work presents an approach to build a high-performance,low-viscous and replaceable separation matrix,semi-crosslinked polyacrylamide(semi-CPA)capillary gel electrophoresis.Non-denatured basic proteins,such as lysozyme,cytochrome C,ribonuclease A and trypsin were separated.The impacts of monomer and cross-linker concentrations on protein separation were studied,and the ability of dynamic capillary inner wall coating was demonstrated.The UV absorption interference by semi-CPA gel matrix was successfully overcome by apartial filling technique,which results in sensitivity 20 times higher than other protein separation method.The excellent separation ability,reproducibility and dynamic coating ability made semi-CPA an ideal separation media in both capillary electrophoresis and microfluidic chip separation scheme.

Studies on Human Immunoglobulin G from GBS Patient (Ⅲ)-The Determination of Molecular Weight of Human Immunoglobulin G by Capillary SDS Gel Electrophoresis

IntroductionGuillain-Barresyndrome(GBS)isconsideredtobeanautoimmunedisorderofperipheralnervoussystem'.Thebalanceofsympatheticnervesandparasympatheticnervesisdisturbed,whichleadstocompleteparalysisi'2'3.Moslpatientsshowthetypicalsymptomsafterbeinginfe...

Conformer Analysis of a Biological G-Quadruplex Element in the Human c-MYC Promoter by Native Polyacrylamide Gel Electrophoresis

The G-quadruplexes undergo complex folding and conformation exchanges.G-quadruplex stability is substantially influenced by sequence,bufer and temperature.Mutational analysis together with nuclear magnetic resonance spectroscopy(NMR),X-ray crystallography and circular dichroism spectroscopy has been proved to be a powerful approach for G-quadruplex structural analysis.Herein,we used DNA sequence mutation and native polyacrylamide gel electrophoresis to investigate the topology and conformations of a G-quadruplex model molecule Pu18 found in the human c-MYC promoter.The guanines(G6,G9 or G18)which were not contributable to G-tetrad formation in c-MYC Pu18 sequence were mutated to thymine or adenine.We screened the buffer and temperature of gel electrophoresis for Pu18.Gel electrophoresis showed that two of the four conformers of c-MYC Pu18 in 100 mM K+buffer were resolved,which was in accordance with the conformations as determined by the 1 H NMR spectra in previous studies.This technique is expected as a general methodology for its easy operation and low cost to facilitate uncovering more yet unidentified G-quadruplex folds and functions,with the assistance of other analytical methods like NMR,X-ray crystallography and circular dichroism spectroscopy.

Establishment of two-dimensional gel electrophoresis for soybean protein isolate and its application

To optimize the conditions for the establishment of two-dimensional gel electrophoresis(2-DE)of soybean protein isolate(SPI),we investigated Ampholine mixture,anodic and cathodic electrolytes,loading amount of sample,acrylamide concentration,p H gradient and gel staining method in twodimensional gel electrophoresis to optimize the protein imaging conditions in two-dimensional gel.The results of mixed-level design experiments showed that Ampholine,loading amount and gel staining method had significant effect(P<0.05)on 2-DE of SPI.The optimal conditions were Ampholine mixture(pH 3–10+pH 5–7 or pH 4–6+pH 5–7),loading amount of 2 mg sample and silver staining.Although the acrylamide concentration of the gel,the p H gradient,the anodic and cathodic electrolyte solutions had significant statistical effects on the protein separation degree,the complexity of the protein composition and the visibility of the gel images were more inclined to the 12%gel,the 3–10 pH gradient and the H3PO4/NaOH electrolyte.According to the established conditions,the hydrolyzed products of SPI emulsion were determined by 2-DE,and the dynamic changes of protein in the process of enzymatic hydrolysis were described.

Gel Electrophoresis of Proteins for the Identification of Crop Varieties

With the development of the international trade and agricultural science and technology, especially after the execution of the rules on protection of new plant varieties, considerable emphasis has been placed on variety identification. Many evidences have suggested that gel electrophoresis have great influence on this area. This paper reviewed study status of various gel electrophoresis, including development of the methods, comparison of these techniques, influence factors, practical applications, achievements obtained and aspects in the future study. With the wider range on protection of new plant varieties in China, electrophoresis will play a more important role in variety identification.

Improvement of Two-Dimensional Gel Electrophoresis for Study of Corm Formation Related Proteins in vitro from Taro (Colocasia esculenta)

Efficient and reproducible sample preparation prior to 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) is a critical step in achieving accurate and reliable data. In this paper, we described a method to prepare protein samples of taro that was compatible with subsequent analysis using 2D-PAGE. We compared proteins from shoot basal region from 0 d and 2 d after the beginning of tuberization. By this method we got about (2 000) spots and high reproducibility. Additionally some changes of protein expression were found.

Differential analysis of two-dimensional gel electrophoresis profiles of spermatozoa protein in human normal motility sperm and idiopathic asthenospermia

Objective: To evaluate the application of two-dimensional electrophoresis in the research of differentially expressed proteins in the human asthenospermia. Methods: Two-dimensional gel electrophoresis was performed on 4 normal sperm samples from healthy men and 4 sperm samples from 4 asthenospermia patients. After silver staining, the differential expression proteins were analyzed by PDQuest 2D analysis software. Results: Six differential protein spots were identified. Four spots showed increased expression in the control gels compared with the patient gels. Conclusion: The protein profiles of differential expression between the normal spermatozoa and idiopathic asthenospermia were established and some differential proteins were found. The data of this study would establish the better fundament for further isolation and identification of differentially expressed proteins in human asthenospermia sperm.

Specific protein expression in a rat model of early focal cerebral ischemia:Fluorescent two-dimensional difference gel electrophoresis

BACKGROUND: The use of fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) has been shown to compensate for the shortcomings of conventional two-dimensional gel electrophoresis, such as poor repeatability and large systematic errors. However, little information is presently available regarding the use of 2D-DIGE to investigate mechanisms of ischemic cerebrovascular diseases. Plasma and body fluids have been utilized in proteomic technology to study ischemic cerebrovascular diseases. OBJECTIVE: To perform proteomic analysis of fresh rat brain tissue in peripheral ischemic regions using 2D-DIGE 6 hours after middle cerebral artery occlusion (MCAO), and to identify specific proteins closely associated with early ischemic cerebrovascular diseases. DESIGN, TIME AND SETTING: Proteomics-based, randomized, controlled, animal experiment was performed at the Laboratories of Neurology and Proteomics, Jilin University between January and April 2006. MATERIALS: 2, 3, 5-triphenyl tetrazolium chloride was purchased from Sigma, USA. Ettan DALTSix system, DeCyder DIA V5.0 differential analysis software, and Ettan matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) were purchased from Amersham Bioscience, Sweden. METHODS: Eight healthy, male, Wistar rats were randomized to experimental and control groups, with four rats in each group. In the experimental group, rat models of focal cerebral ischemia were established by MCAO. In the control group, the internal and external carotid arteries were exposed and then immediately sutured, and the remaining procedures were identical to the experimental group. MAIN OUTCOME MEASURES: At 6 hours after cerebral ischemia, protein expression in the peripheral ischemia region of the experimental group was compared with the control group using 2D-DIGE. Protein spots that exhibited statistical differences between experimental and control groups with > 1.4 attributable risk were screened using DeCyder DIA V5.0 differential analysis software. Differential proteins were identified using MALDI-TOF-MS. RESULTS: Triphenyl tetrazolium chloride staining results revealed pink, normal brain tissue and white, ischemic brain tissue, suggesting successful MCAO establishment. The average matching rate of four 2D-DIGE gels was 92.4%. There were (1 758 ± 43) protein spots on each gel, with similar distribution modes. At 6 hours after focal cerebral ischemia, 13 protein spots exhibited marked expression changes, including significantly increased (n = 7) and decreased (n = 6) expression (P < 0.05). MALDI-TOF-MS results revealed two differential protein spots: α-tubulin and heat shock protein 27, which were significantly decreased in the experimental group compared with the control group (P < 0.05). CONCLUSION: Thirteen protein spots with expression changes were revealed by 2D-DIGE proteomics technology. Of them, α-tubulin and heat shock protein 27 expressions were markedly decreased during the early stage of cerebral ischemia. These two proteins were presumed to be proteins associated with early ischemic cerebrovascular diseases.

A Rapid Electrophoresis Method on Agarose Gel to Characterise Dairy Protein Aggregates

Heat treatment of milk may cause whey proteins and caseins to form aggregates. These soluble and micellar aggregates and their other properties (size, composition, shape, etc.) can affect the techno-functionalities to the milk, conferring interesting or negative features depending on the application in dairy industries. In this study, we propose a new approach to characterise those protein aggregates. SDS-agarose electrophoresis is followed by the calculation of a retention factor (Rf) for each protein spot. Rf allows milk aggregates to be compared qualitatively under the same conditions. This method could be transposed to the dairy industry for a better knowledge of the milk subsequent to heat treatment.

Semiautomatic detection of lanes and bands in DNA gel electrophoresis images

The following article has been retracted due to the investigation of complaints received against it. The Editorial Board found that the same contents have been published in another journal at the same time. The scientific community takes a very strong view on this matter, and the Journal of Biomedical Science and Engineering treats all unethical behavior such as plagiarism seriously. This paper published in Vol.6 No.1 76-84, 2013 has been removed from this site. Title: Semiautomatic detection of lanes and bands in DNA gel electrophoresis images Authors: Ashraf K. Helmy, Ghada S.

Optimization of Two-Dimensional Gel Electrophoresis for Kenaf Leaf Proteins

To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis (2-DE) analysis in kenaf leaf tissues, three extraction methods (trichloroacetic acid/acetone, urea/thiourea, and phenol extraction methods) were applied to the extraction of kenaf leaf protein. The results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 2-DE gels. Furthermore, the 2-DE system was optimized for four aspects: the pH range of IPG (immobilized pH gradient) stripes, sampling methods, sample volumes, and concentration of polyacrylamide gels. The data presented showed that the phenol extraction method is the best method to perform 2-DE analysis of kenaf leaf protein. The protein extracted from phenol extraction method reached the purity of (26.40±0.859)%, showed (25.67±1.53) protein bands in one dimension SDS-PAGE gels, and (1 374±54.44) protein spots on 2-DE gels. The research also indicates that kenaf leaf protein spots were distributed mainly within the pH range of 4-8. More clear background with a better distribution effect and many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading, 24 cm IPG strips (linear pH gradient of 4-7), 1.4 mg samples, and 12% SDS-PAGE gels.

Characterization of YSZ Ceramic Nanopowders Synthesized at Different Temperatures via Polyacrylamide Gel Method

Tetragonal zirconia (T-ZrO2) ceramic nanopowders stabilized with 3 mol% Y2O3 were synthesized via polyacrylamide gel method, using ZrOCl2?8H2O and Y(NO3)3?6H2O as raw materials. The effect of temperature on phase composition and morphology of YSZ nanopowders and sintering behavior of YSZ ceramics was investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM) and Vickers hardness tester. The aging-resistance of YSZ ceramics was measured by means of aging experiments. The results demonstrated that the phase composition of YSZ ceramic nanopowders had no obvious change and it was composed of T-ZrO2. Particle size of well-dispersed YSZ ceramic nanopowders increased from 17 to 35 nm with increasing calcining temperature from 600 to 800 ℃. There was noticeable negative correlation between calcining temperature and the relative density of YSZ ceramic at the same sintering temperature. The aging experiments showed that water vapor facilitated tetragonal to monoclinic phase transformation, and the sample that had smaller grain size exhibited better aging-resistance. It can be concluded that when the calcining temperature is 600 ℃ and sintering temperature is 1550 ℃, the relative density and hardness of YSZ ceramic arrive at the peak of 96.64% and 11.135 GPa respectively, and it has less microcracks and excellent aging-resistance.

Comparative Proteomic Analysis of B. henselae Houston and B. henselae Marseille by Two-dimensional Gel Electrophoresis

Objective To compare the protein difference between B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis. Method Protein samples were prepared by vorterx, ultrasonic treatment, and centrifugation. Protein concentrations were determined by Bradford method. Protein difference was compared by the first IEF and the second SDS-polyacrylamide gel electrophoresis. Results Protein concentrations in samples of Bartonella henselae Houston and Bartonella henselae Marseille were 2.117 μg/μL and 2.200 μg/μL respectively. Sample protein of 40 μg for IPG strips loading was perfect. The results of 2-DE in pH 4 to 7 IPG strips showed that the total protein spots of Bartonella henselae Houston and Bartonella henselae Marseille were 375 and 379 respectively, 95% of the spots were the same between the two strains of Bartonella henselae. Conclusion The procedure of 2-DE may prove successful for the proteomic analysis of Bartonella henselae. Bartonella henselae Houston and Bartonella henselae Marseille are different genotypes.

Synthesis,Characterization and Electromagnetic Studies on Nanocrystalline Nickel Zinc Ferrite by Polyacrylamide Gel

Nanocrystalline 镍锌铁酸盐粉末(Ni_xZni_(1-x ) Fe2O4，为 x=0 的 A，为 x=0.2 的 B，为 x=0.5 的 C，为 x-0.8 的 D 和为 x=1 的 E ) 被聚丙烯酰氨综合胶化方法。X 光检查衍射(XRD ) ，传播电子显微镜学(TEM ) 和波导被用来描绘作文。弄干的胶化粉末是非结晶的 XRD 结果表演，和特征尖晶石Ni_( 0.5 )Zn_( 0.5 ) Fe2O4 达到顶点在胶化为锻烧的温度是的 1 h.When 在 400 °C 被锻烧以后，出现 600 和 800 °C ，平均谷物尺寸被是 10 和 30 nm 的 TEM 识别， respectively.The Ni_xZn_( 1-x ) Fe2O4 粉末在Ni^(2+)离子的增加满足的 8.2-11.0GHz .With 的频率范围有绝缘的损失和磁性的损失，当时，绝缘的参数(epsilon')和Ni_xZn_( 1-x ) Fe2O4 粉末的渗透(mu')减少绝缘的损失( epsilon “)，磁性的损失(亩”)并且思考损失增加。

Crystallization, morphology and luminescent properties of YAG:Ce^(3+) phosphor powder prepared by polyacrylamide gel method

A novel synthesis process, based on the polyacrylamide gel method, was used to prepare Ce-doped YAG phosphor powders. Effects of heat treatment parameters, temperature and holding time, the fluxes, and atmosphere on microstructure and particle morphology as well as luminescent properties of YAG:Ce3+ phosphor powders were studied by X-ray powder diffractometry, scanning electron microscopy, and fluorescence spectrophotometry. The results show that the formation temperature (1 000 ℃) of pure YAG phase is significant low when being synthesized by the polyacrylamide gel method, compared with solid-state reaction. For luminescent properties, the intensity of emission of YAG:Ce3+ phosphor increases steadily with increasing temperature from 900 ℃ to 1 300 ℃ and prolonging holding time from 100 min to 400 min. But blue shift phenomenon is observed for 400 min calcination. Fluxes as BaF2 and H3BO3 can enhance the intensity of emission of phosphor due to the improvement of crystallization of YAG and the stabilization of trivalence cerium ion in YAG host lattice at high temperature. Weak reduction atmosphere can contribute to improvement of the emission intensity of YAG:Ce3+ phosphor powders.

Synthesis,Characterization and Electromagnetic Studies on Nanocrystalline Co_(0.5)Zn_(0.5)Fe_2O_4 Synthesized by Polyacrylamide Gel

Nanocrystalline Co_(0.5)Zn_(0.5)Fe_2O_4 ferrite was synthesized by polyacrylamide gel method.The electromagnetic and microwave absorption properties of the ferrite were investigated.The results indicated that calcining temperature of the ferrite had a significant influence on the effective properties of the ferrite.When the calcining temperature was 500,600 and 700℃,the average size of particles was 10,30 and 80 nm,respectively. The dielectric loss(ε″)and magnetic loss(u″)of the ferrite was around 0.65 and 0.29 at 8.2 GHz,respectively. Microwave absorption properties of the ferrites were simultaneously influenced due to the strong correlation between reflection loss and electromagnetic parameters of the ferrite.