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.

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.

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.

Prevalence and Study of the Clonality of Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Strains in Neonatology at the University Hospitals of Abidjan by the Pulsed Field Gel Electrophoresis and the Quantitative Antibiogram

Background: ESBL-producing strains of Klebsiella pneumoniae, one of the main causes of nosocomial and hospital-acquired infections, are commonly associated with therapeutic impasses. Surveillance of these multidrug-resistant pathogens is a crucial tool for controlling and preventing infections. This surveillance involves the use of appropriate molecular and phenotypic typing techniques. The choice of techniques is based on criteria such as discriminatory power, intra- and inter-laboratory reproducibility, epidemiological concordance, ease of use and cost. The aim of our study was to identify clusters of Extended-Spectrum Beta-Lactamase-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) strains circulating in neonatology using quantitative antibiogram (QA) and Pulsed Field Gel Electrophoresis (PFGE). Materials and Methods: This cross-sectional study included 55 K. pneumoniae strains isolated from a total of 513 samples. These various samples are taken from newborns, healthcare personnel, and the environment. K. pneumoniae identification followed standard bacteriological procedures and was confirmed using the Vitek® 2 (bioMérieux). The detection of the ESBL phenotype was performed using the synergy test. QA and PFGE were used to identify clonal relationships between the various strains isolated. Concordance between these two methods was assessed by calculating Cohen’s KAPPA coefficient and Simpson’s diversity index. Results: Among the 55 K. pneumoniae strains included in this study, 58.2% (32/55) were found to be Extended-Spectrum Beta-Lactamase (ESBL) producers. Most of these strains were isolated from neonatal samples (blood samples and rectal swabs). The quantitative antibiogram method applied to 28 out of the 32 ESBL-producing strains revealed that the isolates were grouped into 5 clusters. Pulsed Field Gel Electrophoresis performed on a total of 16 ESBL-producing strains showed the existence of four profiles. A perfect concordance was observed between the two methods. Conclusion: The results of this study highlighted the existence of clonal strains of various origins within neonatology units.

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 separa- ted. 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 interfer- ence by semi-CPA gel matrix was successfully overcome by a partial 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 capillar- y electrophoresis and microfluidic chip separation scheme.

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.

Improvement of Polyacrylamide Gel Electrophorus in Phytophthora infeatans SSR Marker

[Objective] The paper was to improve polyacrylamide gel electrophorus in Phytophthora infeatans SSR Marker.[Method] With the disease sample of P.infeatans collected from Guyuan in Ningxia Province in 2009 as test material,its DNA was extracted and amplified with PCR,and its products were carried out polyacrylamide gel electrophoresis.[Result] 12% polyacrylamide gel electrophoresis was used to detect primers D13,G11 and PI02,and 8% polyacrylamide gel electrophoresis was used to detect primers PI4B,PI63,SSR4,SSR8 and SSR11,then 0.1% silver nitrate was used to stain,and an ideal electrophoresis and staining effect was obtained.[Conclusion] The electrophoresis and staining method suitable for P.infeatans SSR Marker established in the study had the characteristics of high sensitivity,simple operation and clear bands,which was an effective,simple and quick detection method.

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.

Isolation and purification of guinea pig inner ear antigens by preparative polyacrylamide gel electrophoresis

Objective To isolate and purify the subcomponents of guinea pig inner ear antigens for further study on the autoimmunity of the inner ear.Methods Inner ear tissues were homogenized in phosphate buffered saline containing 0.1% SDS, and then frozen and defrosted repeatedly to extract inner ear antigens. Preparative polyacrylamide gel electrophoresis was used to separate the subcomponents of inner ear antigens. Following electrophoresis,the protein bands were localized by rapid staining and destaining.Results The major protein bands were clearly distinct when 3 mg of crude inner ear antigens was loaded,and the three major subcomponents (31, 42- 45 and 60 kD proteins) accounted for about 25.99%,21.91% and 21.10%, respectively.Conclusion Preparative polyacrylamide gel electrophoresis can be used to purify the major subcomponents of inner ear antigens.

Optimization of Multiplex PCR and Multiplex Gel Electrophoresis in Sunflower SSR Analysis Using Infrared Fluorescence and Tailed Primers

In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis.

A green method of staining DNA in polyacrylamide gel electrophoresis based on fluorescent copper nanoclusters synthesized in situ

The safety of nucleic acid staining dyes has long been recognized to be a problem. Extensive efforts have been made to search for alternatives to the most popular but toxic staining dye, ethidium bromide （EtBr）. However, so far no staining method that can be guaranteed to be suffidently safe has been developed. In this paper, we report a green staining method of DNA in polyacrylamide gel electrophoresis, where in situ synthesis of DNA-templated fluorescent copper nanoclusters （CuNCs） in the gel is achieved to make the DNA bands visible under UV light. Moreover, a comprehensive study of the performance of this staining method has been conducted and the experimental results show that it has favorable sensitivity, stability, and usability. Meanwhile, in our animal experiments, the two reagents （copper sulfate and ascorbic acid） as well as the synthesized CuNCs have been proven to be non-toxic in contact with skin. In addition, all the reagents employed in this work are readily available and low cost, and the procedure is simple to carry out. Therefore, this novel staining method based on the in situ synthesis DNA-templated fluorescent CuNCs has many potential applications.

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.

Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.

Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments

The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter （OM）, total nitrogen （TN）, total phosphorus （TP）, pH and redox potential （Eh）. Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341 f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis （DGGE）. The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. and some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core.

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

Guillain-Barre syndrome (GBS) is considered to be an autoimmune disorder of peripheralnervous system. In this paper. capillary SDS gel electrophoresis was performed on neutral coatedfused-silica capillary to determine the molecular weight of purified IgG samples from GBS patient.

Synthesis of YAG∶Ce^(3+) Phosphor by Polyacrylamide Gel Method and Promoting Action of α-Al_2O_3 Seed Crystal on Phase Formation

YAG： Ce^3 ＋ phosphor particles were prepared using polyacrylamide gel method. The structure evolution of powders during annealing process was followed by X-ray diffraction determination. It is found that some intermediate phases, including θ-Al2O3, YAM and YAP, are formed when calcining polyacrylamide gel, however, the pure YAG phase can be formed directly when calcining polyacrylamide gel with α-Al2O3 as seed crystal. These facts show that the existence of α- Al2O3 seed crystal can block the formation of θ-Al2O3, YAM and YAP, and accelerate its reaction with Y2O3 to form YAG phase directly at lower temperature. The emission peak of prepared YAG ： Ce^3 ＋ phosphor is wide with maximum at 550 nm and the exitation band has two peaks, the major one is around at 460 nm, which matches the blue emission of GaN LED and is suitable for the assemble of white LED. Some fluxes can enhance the photoluminescence intensity of phosphor particles, that can be attributed both to the improvement of crystallization processes of YAG and to the stabilization of trivalence cerium ion in YAG：Ce^3 ＋.

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

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 nickel zinc ferrite powders （Ni=Zn1-xFe2O4, A for x=0, B for x=0.2, C for x=0.5, D for x= 0.8 and E for x= 1） were synthesized by polyacrylamide gel method. X-ray diffraction （XRD）, transmission electron microscopy （TEM） and wave-guide were used to characterize the composition. The XRD results show that the dried gel powders are amorphous, and the characteristic peaks of the spinel Ni0.5Zn0.5Fe2O4 appear after the gel is calcined at 400℃ for 1 h. When the calcining temperatures are 600 and 800℃, the average grain sizes are identified by TEM to be 10 and 30 nm, respectively. The NixZn1-xFe2O4 powders have both dielectric loss and magnetic loss in the frequency range of 8.2-11.0GHz. With the increase of Ni^2＋ ions content, the dielectric parameters （ε′） and permeability （u′） of the NixZn1-xFe2O4 powders decrease while the dielectric loss （ε″）, magnetic loss （u″） and the reflection loss increase.

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

Nanocrystalline Co0.5Zn0.5Fe2O4 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 （μ″） 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.

Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5～6.2, 4.6,5.1,5.5～5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.