• Preventive Nutrition and Food Science
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• v.13 no.4
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• pp.366-369
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• 2008

Proteins secreted from bifidobacteria are believed to play important roles in human intestines via interacting with different host cells. In this respect, proteins secreted from Bifidobacterium pseudocatanulatum BP1, which has been rarely studied, were analyzed by two-dimensional gel electrophoresis (2DE). Using this approach, approx-imately 21 protein spots on a 2DE gel were detected and 10 of these spots were identified by mass spectrometry. Five spots were identified as hypothetical proteins and the remaining 5 spots were identified as a putative iron-side-rophore binding lipoprotein, a short-chain dehydrogenase/reductase SDR, an exonuclease, cytochrome P450 hydroxylase, and a putative dehydrogenase. The identification of secreted putative iron-siderophore binding lipoprotein was highly interesting since it is an important protein that is involved in ferric iron uptake in pathogenic bacteria. This finding could accelerate studies on the probiotic effect of Bifidobacterium by explaining the competition between bifidobacteria and intestinal pathogens for ferric iron.

• BMB Reports
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• v.32 no.5
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• pp.445-450
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• 1999

We purified and characterized a bovine pregnancy-associated protein in pregnant cow urine using two-dimensional gel electrophoresis. Urine from cows was collected according to their status of pregnancy and non-pregnancy. Proteins in the cow urine were fractionated with 50% ammonium sulfate prior to two-dimensional gel electrophoresis. Proteins separated on the gels were compared in terms of expression level and new expression by molecular mass and isoelectric point. We localized two pregnancy-associated protein spots on the gels at molecular masses of 24 kDa and 20 kDa and isoelectric points of 5.5 and 5.7, respectively. Likewise, two non-pregnancy specific proteins were localized at 27 kDa and 28 kDa with isoelectric points of 5.7 and 5.9, respectively. To rule out the possibility that environmental or genetic factors might influence the expression of the proteins, we demonstrated the pregnancy-associated expression of the proteins in two-dimensional gels with pregnant urine taken from cows raised in a different institute. The pregnancy-associated protein with molecular mass of 20 kDa and isoelectric point of 5.7, namely spot 2, was microsequenced and found to be highly homologous to the bovine collagen alpha 1 chain.

• Bulletin of the Korean Chemical Society
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• v.23 no.11
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• pp.1511-1512
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• 2002

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.25-31
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• 2006

Changes of CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in the glyphosate-tolerant Roundup Ready soybean were examined using purified CP4 EPSPS produced in cloned Escherichia coli as a control. CP4 EPSPS in genetically modified soybean was detected by twodimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) with databases. CP4 EPSPS in soybean products was resolved on 2-DE by first isoelectric focusing (IEF) based on its characteristic pI of 5.1, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) based on its molecular mass of 47.5 kDa. We quantified various percentages of soybean CP4 EPSPS. The quantitative analysis was performed using a 2D software program on artificial gels with spots varying in Gaussian volumes. These results suggested that 2-DE image analysis could be used for quantitative detection of GM soybean, unlike Western blotting.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.58 no.4
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• pp.388-392
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• 2013

The proteins from functional rice cultivars (Nogwonchalbyeo, Giant embryonic, Arhyangchalbyeo, and Goamibyeo) and general white rice were extracted and separated using two-dimensional (2D) gel electrophoresis. A wide variation in the molecular weight (MW) and pH range of the expressed proteins in rice samples were observed. The green-kerneled rice (Nogwonchalbyeo) exhibited proteins with MW of 9-57 kDa and appeared at a pH range of 4-7. The Giant embryonic contained proteins with MW of 31-63 kDa and a pH range of 5-6. The aromatic glutinous rice (Arhyangchalbyeo) showed proteins with MW of 24-28 and pH of 5.8-6.8. The high-amylose rice (Goamibyeo) exhibited proteins with MW of 3-63 and pH of 5.2-5.6. The identified proteins uniquely found and highly expressed in each cultivar may have a significant role on rice functionality. The results illustrate that the 2D gel electrophoresis is a valuable method in the determination of the protein expression profiles in functional rice grains and may be useful in the identification of specific marker proteins associated with the functional property of rice.

• Journal of Microbiology and Biotechnology
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• v.22 no.5
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• pp.654-658
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• 2012

Osteoarthritis (OA) is the most common rheumatic pathology. One of the major objectives of OA research is the development of early diagnostic strategies such as those using proteomic technology. Synovial fluid (SF) in OA patients is a potential source of biomarkers for OA. The efficient and reliable preparation of SF proteomes is a critical step towards biomarker discovery. In this study, we have optimized a pretreatment method for twodimensional gel electrophoresis (2DE) separation of the SF proteome, by enriching low-abundance proteins and simultaneously removing hyaluronic acid, albumin, and IgG. SF samples pretreated using this optimized method were then evaluated by 1DE and 2DE separation followed by immunodetection of cartilage oligomeric matrix protein (COMP), a known OA biomarker, and by the identification of 3 proteins (apolipoprotein, haptoglobin precursor, and fibrinogen D fragment) that are related to joint diseases.

• Applied Biological Chemistry
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• v.30 no.2
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• pp.163-168
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• 1987

A method, based upon the separation of cellular proteins by one-and two-dimensional electrophoresis was used for distinguishing butween Bradyrhizobium japonicum strains and Rhizobium fredii strains. Significant differences in protein pattern of one-dimensional SDS-PAGE vs-ere observed between Rhizobium fredii strains and Bradyrhizobium japonicum strains. The differences in six distinct main lands were observed among total 52 kinds of protein bands. Furthermore, the distribution of proteins in two groups by two-dimensional polyacrylamide gel electrophoresis was very different. The majority of visible proteins of Rhizobium fredii were acidic, whereas those of Bradyrhizobium japonicum were basic. In addition, amino acid composition was analyzed to detect the differences between two groups. No significant differences in amino acid composition were observed between Bradyrhizobium japonicum strains and Rhizobium fredii strains. The results indicate that one-and two-dimensional polyacrylamide gel electrophoresis were useful for identifying rhizobia isolates. One-dimensional SDS-PAGE of rhizobia proteins provided a rapid method for screening a large number of isolates, whereas two-dimensional electrophoresis was more of resolution and easiness for analyzing protein spots.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.89-95
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• 2007

Improvements in the dissolution of proteins in two-dimensional gel electrophoresis have greatly advanced the ability to analyze the proteomes of microorganisms under a wide variety of physiological conditions. This study examined the effect of various combinations of chaotropic agents, a reducing agent, and a detergent on the dissolution of the Streptomyces peucetius cytosolic proteins. The use of urea alone in a rehydration buffer as a chaotropic agent gave the proteome a higher solubility than any of the urea and thiourea combinations, and produced the highest resolution and clearest background in two-dimensional gel electrophoresis. Two % CHAPS, as a detergent in a rehydration buffer, improved the protein solubility. After examining the effect of several concentrations of reducing agent, 50 mM DTT in a rehydration buffer was found to be an optimal condition for the proteome analysis of Streptomyces. Using this optimized buffer condition, more than 2,000 distinct and differentially expressed soluble proteins could be resolved using two-dimensional gel electrophoresis with a pI ranging from 4-7. Under this optimized condition, 15 novel small proteins with low-level expression, which could not be analyzed under the non-optimized conditions, were identified. Overall, the optimized condition helped produce a better reference gel for Streptomyces peucetius.

• Genomics & Informatics
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• v.9 no.4
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• pp.197-199
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• 2011

The biological interpretation of two-dimensional (2D) gel electrophoresis experiments is a key step toward understanding the functions of biological systems. We here present a web-based integrated database, called 2DSpotDB, for the management of proteome data derived from several pathogens. The 2DSpotDB was established as a part of the management of a pathogen proteome project at the Korea National Institute of Health. The goals of the 2DSpotDB implementation are to store and define important pathogen genes, retrieve information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry, and create an integrated system to provide pathogen proteome information for biological scientists. This database currently contains 14 gels and information on 387 protein spots, among which 329 proteins were identified and annotated.

• BMB Reports
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• v.39 no.2
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• pp.216-222
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• 2006

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT(dithiothreitol) and 0.2% carrier ampholytes; (b) 5M urea, 2M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), 40mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7M urea, 2M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.