Identification of differentially expressed proteins in human stage Ⅰ lung adenocarcinoma and tumor-adjacent tissues with two-dimension gel electrophoresis profiling

Objective： The aim of this study was to establish reproducible two-dimensional electrophoretic assay used for profiling and identification of differentially expressed proteins in human stage I lung adenocarcinoma and paired normal tumor-adjacent tissue. Methods： The proteins from 12 human stage I lung adenocarcinoma tissues and normal tumor-adjacent tissues were separated using isoelectric focusing electrophoresis （the first dimension） and the subsequent homogeneous SDS-polyacrylamide gel electrophoresis （SDS-PAGE） （the second dimension）. The differentially expressed proteins were determined with PDQuest image analysis software, and identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS） and database searching. Results： The well-reproducible 2-DE gel patterns of human stage I lung adenocarcinoma and normal tumor-adjacent tissues were profiled and 26 differentially expressed proteins uncovered. Nine of these 26 protein spots were cut out from the preparation gels and determined with MALDI-TOF-MS. Searching against the protein database, four candidate proteins were identified. They were 60S acidic ribosomal protein P2, Cathepsin B1, Apolipoprotein A-I precursor, and La 4.1 protein. Conclusion： In this study, high reproducible 2-DE gel protein images of human stage I lung adenocarcinoma and paired normal tumor-adjacent tissues were achieved successfully, and 4 differentially expressed proteins were revealed. These data will be helpful for screen of early biomarker and study of molecular mechanisms of human lung adenocarcinoma.

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.

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.

Two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry for detection of protein expression profiles in the hippocampus following closed brain injury

Gene expression profile changes in brain regions following traumatic brain injury at the gene level cannot sufficiently elucidate gene expression time, expression amount, protein post-translational processing or modification. Therefore, it is necessary to quantitatively analyze the gene expression profile using proteomic techniques. In the present study, we established a rat model of closed brain injury using Marmarou＇s weight-drop device, and investigated hippocampal differential protein expression using two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry. A total of 364 protein peaks were detected on weak cation exchange-2 protein chips, including 37 differential protein peaks. 345 protein peaks were detected on immobilized metal affinity capture arrays-Cu, including 12 differential protein peaks Further examination of these differential proteins revealed that glucose-regulated protein and proteasome subunit alpha type 3 expression were significantly upregulated post-injury. These results indicate that brain injury can alter protein expression in the hippocampus, and that glucose-regulated protein and proteasome subunit alpha type 3 are closely associated with the occurrence and development of traumatic brain injury.

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.

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.

In Vitro Protein Expression Profile of Campylobacter jejuni Strain NCTC11168 by Two-dimensional Gel Electrophoresis and Mass Spectrometry

Objective To investigate the protein expression profiles of the major food‐borne pathogen Campylobacter jejuni NCTC11168.Methods Membrane and soluble cellular proteins were extracted from the genome‐sequenced C.jejuni strain NCTC11168.Protein expression profiles were determined using two‐dimensional gel electrophoresis（2‐DE）.All the detected spots on the 2‐DE map were subjected to matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry（MALDI‐TOF/TOF） analysis.Results A total of 537 and 333 spots were detected from the whole cell and membrane‐associated proteins of C.jejuni NCTC11168 cultured on Columbia agar medium at 42 ℃ by 2‐DE and Coomassie Brilliant Blue staining,respectively.Analyses of whole cell and membrane‐associated proteins included 399 and 133 spots,respectively,which included 182 and 53 functional proteins identified by MALDI‐TOF/TOF analysis.Conclusion The comprehensive expression protein profiles of C.jeuni NCTC11168 obtained in this study will be useful for elucidating the roles of these proteins in further pathogenesis investigation.

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.

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.

Protein expression of sensory and motor nerves Two-dimensional gel electrophoresis and mass spectrometry

The present study utilized samples from bilateral motor branches of the femoral nerve, as well as saphenous nerves, ventral roots, and dorsal roots of the spinal cord, to detect differential protein expression using two-dimensional gel electrophoresis and nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry techniques. A mass spectrum was identified using the Mascot search. Results revealed differential expression of 11 proteins, including transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an unnamed protein product （gil55628）, gfyceraldehyde-3-phosphate dehydrogenase-like protein, lactoylgfutathione lyase, adenyfate kinase isozyme 1, two unnamed proteins products （gil55628 and gi11334163）, and poly（rC）-binding protein 1 in motor and sensory nerves. Results suggested that these proteins played roles in specific nerve regeneration following peripheral nerve injury and served as specific markers for motor and sensory nerves.

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： a-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, a-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.

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.

Cerebrospinal fluid diagnostic markers for two-dimensional electrophoresis-mass spectrometry in Parkinson’s disease patients

BACKGROUND： Previous studies have confirmed the existence of specific proteins in body fluid of Parkinson＇s disease （PD） patients. However, the existing research has contained several interference factors with poor reproducibility and has not focused on patients grouped according to disease duration. OBJECTIVE： To verify differential expression of proteins in cerebrospinal fluid of PD patients grouped in order of disease severity through the use of two-dimensional electrophoresis-mass spectrometry methods. DESIGN, TIME AND SETTING： The proteomic-based, case-control study was performed between September 2008 and June 2009 at the Key Laboratory of Neurology in the First Affiliated Hospital of Chongqing Medical University. PARTICIPANTS： A total of 52 outpatients and/or inpatients, who were admitted to the Department of Neurology in the First Affiliated Hospital of Chongqing Medical University between 2008 and 2009, were randomized into the present study. Among them, 27 PD patients served as the PD group and were assigned to three subgroups according to modified Webster, Hoehn, and Yahr rating scales： 14 = mild, 8 = moderate, and 5 = severe; non-PD group of 16 patients included 5 cases of viral meningitis, 3 cases of acute myelitis, 1 case of Guillain-Barre syndrome, 2 cases of tuberculous meningitis, 2 cases of restless legs syndrome, and 3 cases of essential tremor; control group （n = 9） consisted of muscular tension headache in 6 cases, as well as syncope, trigeminal neuralgia, idiopathic orthostatic hypotension in 1 case. METHODS： Cerebrospinal fluid was collected from the involved patients using the lumbar puncture method. Proteins in the cerebrospinal fluid were separated by two-dimensional electrophoresis. MAIN OUTCOME MEASURES： Characteristics of protein electrophoresis patterns were analyzed, differentially expressed proteins were detected using matrix-assisted laser desorption ionization time of flight mass spectrometry, and protein data were analyzed in the Mascot database. RESULTS： Five protein electropherograms were analyzed by PDQuest 8.0, and （789 ± 32） protein spots were observed. There were significant differences in four protein spots in each of the PD sub-groups compared with the non-disease and control groups. Expression was down-regulated in three protein spots and up-regulated in one protein spot; 100% repetition rate was observed in four protein spots. According to the Mascot database, protein spots with down-regulated expression were as follows： DNA-guided RNA polymerase III subunit RPC5 （score： 50 points）; double serine, threonine, and tyrosine protein kinase （score： 64 points, P 〈 0.05）; activity-regulated cytoskeleton-associated protein （score： 58 points, P 〈 0.05）. However, G2 mitotic-specific cyclin was up-regulated （score： 84 points, P 〈 0.05）. CONCLUSION： Differential protein expression in the cerebrospinal fluid of PD patients was detected by two-dimensional electrophoresis-mass spectrometry, revealing changes in DNA-guided RNA polymerase III subunit RPC5, double serine, threonine, and tyrosine protein kinase, activity-regulated cytoskeleton-associated protein, and G2 mitotic cell cyclin, with good reproducibility.

Two-dimensional Electrophoresis Analysis of Differential Protein Expression in Squamous Carcinoma of the Cervix

Objective： To establish and optimize the two-dimensional gel electrophoresis （2-DE） maps of squamous carcinoma of the cervix and to study the protein difference between squamous carcinoma of the cervix （SCC） and normal cervical tissue. Methods： Using Two-dimensional gel electrophoresis followed by computer-assisted image analysis, the differential proteins between squamous carcinoma of the cervical tissue and normal cervical tissue were compared. Then using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, the differential proteins were identified. Results： The well-resolved and reproducible two-dimensional gel electrophoresis patterns of squamous carcinoma of the cervix tissue and normal cervical tissue were obtained. After silver staining, the average matching ratio of squamous carcinoma of the cervix was 86.1%. There was a good reproducibility of spot position in 2-DE map, with average deviation in IEF direction of 0.95±0.13 mm, while in SDS-PAGE direction it was 1.20±0.18 mm. Ten protein spots were identified by mass spectrometry, some of which were involved in cell proliferation, cell apoptosis, intracellular enzymes, structural proteins, cycle regulation, and tumor occurrence. Conclusion： The differentially expressed proteins provide a fundamental basis for further study of human squamous carcinoma of the cervix and screening of its specific markers.

Differentially expressed phosphoproteins in diazoxide-pretreated ventricular myocytes by two-dimensional electrophoresis and mass spectrometry in vitro

Objectives To analyze and identify differentially expressed phosphoproteins associated with mitochondrial KATP channel opening. Methods： Adult rat ventricular myocytes were isolated, cultured, and identified, and pretreated without or with 100 μmol/L diazoxide for 10 min. Phosphoproteins prepared and enriched from the control and diazoxide-pretreated cells were separated by two-dimensional gel electrophoresis （2-DE） followed by sliver staining. The obtained interesting phosphoproteins were further identified by mass spectrometry. Results. Associated with diazoxide preconditioning, the proteins of chaperonin containing TCP-1 and hypothetical protein XP-346548 were phosphorylated significantly （P〈0. 01）, while the 94-kDa glucose-regulated protein, calpactin I heavy chain and ferritin were dephosphorylated markedly （P〈0. 01）. Conclusion： These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins in order to respond diazoxide preconditioning, and these phosphorylated protein may mediate the downstream signaling of cardioprotection by mitochondrial KATp channel opening induced by ischemic preconditioning.

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.

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.

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.

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.