In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.
Cordyceps ; chemistry ; Desiccation ; methods ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; analysis ; Molecular Weight

BACKGROUND: We developed skin prick test (SPT) reagents for common inhalant allergens that reflected the real exposure in Korea. The study aim was to evaluate diagnostic usefulness and allergen potency of our inhalant SPT reagents in comparison with commercial products. METHODS: We produced eight common inhalant allergen SPT reagents using total extract (Prolagen): Dermatophagoides farinae, Dermatophagoides pteronyssinus, oak, ragweed, mugwort, Humulus japonicus pollens, as well as cat and dog allergens. We compared the newly developed reagents with three commercially available SPT reagents (Allergopharma, Hollister-Stier, Lofarma). We measured total protein concentrations, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), major allergen concentration, and biological allergen potencies measured by immunoglobulin E (IgE) immunoblotting and ImmunoCAP inhibition test. RESULTS: Diagnostic values of these SPT reagents were expressed as positivity rate and concordance rate of the results from ImmunoCAP allergen-specific IgE test in 94 allergic patients. In vitro analysis showed marked differences in protein concentrations, SDS-PAGE features, major allergen concentrations, and biological allergen potencies of four different SPT reagents. In vivo analysis showed that positive rates and concordance rates of Prolagen® SPT reagents were similar compared to the three commercial SPT reagents. CONCLUSION: The newly developed Prolagen® inhalant SPT reagents are not inferior to the commercially available SPT reagents in allergy diagnosis.
Allergens ; Allergy and Immunology ; Ambrosia ; Animals ; Artemisia ; Cats ; Dermatophagoides farinae ; Dermatophagoides pteronyssinus ; Diagnosis ; Dogs ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Humans ; Humulus ; Hypersensitivity ; Immunoblotting ; Immunoglobulin E ; Immunoglobulins ; In Vitro Techniques* ; Indicators and Reagents* ; Korea ; Methods* ; Pollen ; Skin* ; Sodium

OBJECTIVES: To explore the ability of inhibition resistibility of multiple displacement amplification （MDA） in samples with inhibitors. To explain the application and value of MDA in forensic medicine by comparing with using magnetic beads methods （MBM） to purify sample. METHODS: Different concentrations of hemoglobin and humid acid （HA） mixed with DNA samples and then divided the samples into MDA group, MBM group and control group. D3S1358 locus was amplified and detected by polyacrylamide gel electrophoresis detection system and AmpFℓSTR® Identifiler™ Plus Kit-capillary electrophoresis detection system. RESULTS: When hemoglobin concentrations exceed 1 ng/μL or HA concentrations exceed 0.1 ng/μL, amplification products could not be obtained by single-locus system in control group. When hemoglobin concentration exceeds 100 ng/μL or HA concentrations exceed 1 ng/μL, the samples could not be amplified by MBM. Inhibitors in different concentrations were amplified successfully in MDA group without any influence from inhibitors. CONCLUSIONS MDA has the capability to remove the inhibition of hemoglobin and HA, which is better than MBM and has a certain value in forensic practices.
DNA/analysis* ; Electrophoresis, Polyacrylamide Gel ; Forensic Medicine/methods* ; Hemoglobins ; Humans ; Humic Substances ; Nucleic Acid Amplification Techniques

In proteomic research, to improve protein solubility of membrane proteins and nuclear proteins, buffers containing high concentration of detergent, such as 4% SDS, were widely used. However, high concentration of detergent might severely interfere with the downstream proteomic analysis, including protein quantitation and trypsin digestion. To improve the proteomic compatibility of buffers with high concentration of detergent, we used short gel method to pretreat buffers containing detergent. Protein samples were first separated by a short (2-2.5 mm) SDS-PAGE electrophoresis, and proteins were quantitated by comparing with bovine serum albumin standards via optical density analysis. The gel was then cut and peptides were recovered using in-gel digestion. The quantitative linearity range of this method was 1 to 8 μg. The quantitation was accurate and reproducible. After short gel analysis, recovered peptides generated high mass spectrometry signals. In conclusion, short gel method eliminated the interference of high concentration detergent in the proteomics analysis, and it was suitable for protein samples' pretreatment, and was worth to apply in proteomic research.
Detergents ; chemistry ; Electrophoresis, Polyacrylamide Gel ; methods ; Mass Spectrometry ; Membrane Proteins ; chemistry ; Nuclear Proteins ; chemistry ; Proteins ; chemistry ; Proteomics ; methods ; Trypsin

Despite the development of new technologies, new challenges still remain for large scale proteomic profiling when dealing with complex biological mixtures. Fractionation prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is usually the preferred method to reduce the complexity of any biological sample. In this study, a gel LC-MS/MS approach was used to explore the stage specific proteome of Cryptosporidium (C.) parvum. To accomplish this, the sporozoite protein of C. parvum was first fractionated using SDS-PAGE with subsequent LC-MS/MS analysis. A total of 135 protein hits were recorded from 20 gel slices (from same gel lane), with many hits occurring in more than one band. Excluding all non-Cryptosporidium entries and proteins with multiple hits, 33 separate C. parvum entries were identified during the study. The overall goal of this study was to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins present in lower abundance in a complex protein mixture.
Chemical Fractionation/methods ; Chromatography, Liquid/methods/veterinary ; Cryptosporidium parvum/*chemistry/growth & development/metabolism ; Electrophoresis, Polyacrylamide Gel/methods/veterinary ; Gene Expression Profiling/*methods/veterinary ; Proteome/analysis ; Proteomics/*methods ; Protozoan Proteins/*analysis ; Sporozoites/chemistry/metabolism ; Tandem Mass Spectrometry/methods/veterinary

OBJECTIVES: Fish vitellogenin (VTG) is produced in the female liver during oogenesis through the estradiol cycle and produced in the male liver by endocrine disrupting chemicals (EDCs) such as alkylphenols. In this study, we propose that the VTG concentration in the pale chub could be detected using monoclonal antibodies and polyclonal antibodies against vitellin (Vn) in a VTG enzyme-linked immunosorbent assay (ELISA) system. METHODS: Monoclonal antibodies and polyclonal antibodies were produced using the Vn extracted from the matured ovum of the ovary. The VTG was extracted from the plasma of the male pale chub. The Vn and VTG were confirmed by measuring the molecular weight of their proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of the antibodies was checked through western blotting methods. The assay system was validated with respect to optimal assay concentrations, specificity, recovery, and intra- and inter-assay variations. RESULTS: The Vn consisted of two protein bands with apparent molecular weights of 64 and 37 kDa. The SDS-PAGE indicated protein weights of 146 and 77 kDa in the VTG. The assay range was 15.6 ng/mL to 2,000 ng/mL, and the value of the intra- and inter-assay variations were within 10.0% and 14.7%, respectively. The recovery rate was 99.5+/-5.5%. CONCLUSIONS: A sandwich ELISA was developed that could be used to qualify the VTG of pale chub in screening for EDCs. Pale chub is an ideal species for observing estrogen activity in the environment because of its extensive habitat and extensive food chain. The ELISA developed here would be more favorable than those for other species for determining the effect of long-term food chain accumulation of EDCs in aquatic environments.
Antibodies ; Antibodies, Monoclonal ; Blotting, Western ; Cyprinidae* ; Ecosystem ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Endocrine Disruptors ; Enzyme-Linked Immunosorbent Assay* ; Estradiol ; Estrogens ; Female ; Food Chain ; Humans ; Liver ; Male ; Mass Screening ; Methods ; Molecular Weight ; Oogenesis ; Ovary ; Ovum ; Plasma ; Platypus* ; Sensitivity and Specificity ; Sodium ; Vitellins* ; Vitellogenins* ; Weights and Measures

Base Sequence ; Binding Sites, Antibody ; Biochemistry ; methods ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Genetic Vectors ; Immunoglobulin Fc Fragments ; genetics ; metabolism ; Immunoglobulin G ; genetics ; metabolism ; Ligands ; Molecular Sequence Data ; Plasmids ; Protein Binding ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; Staphylococcal Protein A ; genetics ; metabolism

OBJECTIVETo develop a solid phase PCR method by covalent single point immobilization for recycle utilization of human genome.

METHODSPolymethacrylamide gel was selected as a solid PCR carrier based on DNA-hydrogel copolymer chemistry presented by Mirzabekov. (CH2)6NH2 amino-modified PCR product and randomly fractured formic acid-modified plasmid pGEM-T-HLA-G were used as templates. The specificity of the attachment chemistry was characterized by acrylamide gel electrophoresis, and the thermal stability of method was demonstrated by PCR. This method was applied for the recycle utilization of human genome. Sequencing was used to exclude the possibility of introduced mutations during modification and immobilization procedures.

RESULTSThe PCR detections of plasmid DNA and human genome DNA immobilized by polymethacrylamide gel was successful. The thermal stability of method was successfully demonstrated by performing PCR after 16 rounds of standard 36 PCR cycles. And the sequencing was found no mutation.

CONCLUSIONThe DNA immobilization method with polymethacrylamide gel as a solid phase carrier is stable and specific, which can be a possible approach for realizing recycle utilization of human genome for whole-genome sequencing and SNP detection.

Electrophoresis, Polyacrylamide Gel ; Genome, Human ; Humans ; Hydrogels ; Immobilized Nucleic Acids ; analysis ; Polymerase Chain Reaction ; methods

OBJECTIVETo study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure.

METHODSFP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index.

RESULTSCriteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

CONCLUSIONEstablishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China.

Blotting, Western ; methods ; Borrelia burgdorferi Group ; pathogenicity ; China ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Humans ; Lyme Disease ; diagnosis ; microbiology

OBJECTIVETo establish a stable, rapid multiplex PCR assay combined with PAGE gel electrophoresis for simultaneously detecting FLT3-ITD and NPM1 mutations in acute myeloid leukemia (AML).

METHODSCapillary electrophoresis (CE) and PAGE gel electrophoresis were simultaneously used to analyze FLT3-ITD and NPM1 mutations in 117 de novo AML patients with normal cytogenetic findings.

RESULTSFor certain mutations, the length of mutated double-stranded DNA is longer than wild-type DNA. Since FLT3-mut (420 bp) is longer than FLT3-wt (327-332 bp), and NPM1-mut (172 bp) is longer than NPM1-wt (168 bp), heteroduplex will move more slowly during PAGE gel electrophoresis than homoduplex. Therefore the mutations may be detected. A total of 117 CN-AML patients were analyzed with CE and PAGE gel electrophoresis, and the results were identical, which included 18 (15.4%) patients with FLT3-ITD+/NPM1-, 19 (16.2%) patients with FLT3-ITD+/NPM1+, 25 (21.4%) patients with FLT3-ITD-/NPM1+, and 55 (47.0%) patients with FLT3-ITD-/NPM1-.

CONCLUSIONBoth types of electrophoresis assays may provide a rapid and handy assay for simultaneous detection of FLT3-ITD and NPM1 mutations. CE is relatively sensitive, stable; while PAGE electrophoresis is relatively simple, cheap, and reliable, which may be suitable for primary hospitals and preliminary screening.

Base Sequence ; Electrophoresis, Polyacrylamide Gel ; methods ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Male ; Molecular Sequence Data ; Multiplex Polymerase Chain Reaction ; methods ; Mutation ; Nuclear Proteins ; genetics ; fms-Like Tyrosine Kinase 3 ; genetics