• Title/Summary/Keyword: ProteinChip

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Unfolded Histidine-Tagged Protein is Immobilized to Nitrilotriacetic Acid-Nickel Beads, But Not the Nickel-Coated Glass Slide

  • Cho Min-Ho;Ahn Sun-Young;Park Heon-Yong
    • Genomics & Informatics
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    • v.4 no.3
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    • pp.133-136
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    • 2006
  • The adsorption of proteins on the surface of glass slides is essential for construction of protein chips. Previously, we prepared a nickel-coated plate by the spin-coating method for immobilization of His-tagged proteins. In order to know whether the structural factor is responsible for the immobilization of His-tagged proteins to the nickel-coated glass slide, we executed a series of experiments. First we purified a His-tagged protein after expressing the vector in E. coli BL21 (DE3). Then we obtained the unfolding curve for the His-tagged protein by using guanidine hydrochloride. Fractions unfolded were monitored by internal fluorescence spectroscopy. The ${\Delta}G_{H20}$ for unfolding was $2.27kcal/mol{/pm}0.52$. Then we tested if unfolded His-tagged proteins can be adsorbed to the nickel-coated plate, comparing with $Ni^{2+}-NTA$ (nitrilotriacetic acid) beads. Whereas unfolded His-tagged proteins were adsorbed to $Ni^{2+}-NTA$ beads, they did not bind to the nickel-coated plate. In conclusion, a structural factor is likely to be an important factor for constructing the protein chips, when His-tagged proteins will immobilize to the nickel-coated slides.

Effects of Urea Level and Sodium DL-malate in Concentrate Containing High Cassava Chip on Ruminal Fermentation Efficiency, Microbial Protein Synthesis in Lactating Dairy Cows Raised under Tropical Condition

  • Khampa, S.;Wanapat, Metha;Wachirapakorn, C.;Nontaso, N.;Wattiaux, M.
    • Asian-Australasian Journal of Animal Sciences
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    • v.19 no.6
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    • pp.837-844
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    • 2006
  • Four, lactating dairy cows were randomly assigned according to a $2{\times}2$ Factorial arrangement in a $4{\times}4$ Latin square design to study supplementation of urea level (U) at 2 and 4% and sodium dl-malate (M) at 10 and 20 g/hd/d in concentrate. The treatments were as follows U2M10, U2M20, U4M10 and U4M20, respectively. The cows were offered the treatment concentrate at a ratio to milk yield at 1:2.5 and urea-treated rice straw was fed ad libitum. The results have revealed that rumen fermentation and blood metabolites were similar for all treatments. The populations of protozoa and fungal zoospores were significantly different as affected by urea level and sodium dl-malate. In addition, the viable bacteria were similar for amylolytic and proteolytic bacteria. Cellulolytic bacteria were significantly affected by level of sodium dl-malate especially Selenomonas ruminantium and Megasphaera elsdenii while Butyrivibrio fibrisolvens was significantly affected by level of urea supplementation. In conclusion, the combined use of concentrate containing high level of cassava chip at 75% DM with urea at 4% in concentrate and sodium dl-malate at 20 g/hd/d with UTS as a roughage could improv rumen ecology and microbial protein synthesis efficiency in lactating dairy cows.

Effect of Carbohydrate Sources and Levels of Cotton Seed Meal in Concentrate on Feed Intake, Nutrient Digestibility, Rumen Fermentation and Microbial Protein Synthesis in Young Dairy Bulls

  • Wanapat, Metha;Anantasook, N.;Rowlinson, P.;Pilajun, R.;Gunun, P.
    • Asian-Australasian Journal of Animal Sciences
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    • v.26 no.4
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    • pp.529-536
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    • 2013
  • The objective of this study was to investigate the effect of levels of cottonseed meal with various carbohydrate sources in concentrate on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis in dairy bulls. Four, 6 months old dairy bulls were randomly assigned to receive four dietary treatments according to a $2{\times}2$ factorial arrangement in a $4{\times}4$ Latin square design. Factor A was carbohydrate source; cassava chip (CC) and cassava chip+rice bran in the ratio of 3:1 (CR3:1), and factor B was cotton seed meal levels in the concentrate; 109 g CP/kg (LCM) and 328 g CP/kg (HCM) at similar overall CP levels (490 g CP/kg). Bulls received urea-lime treated rice straw ad libitum and were supplemented with 10 g of concentrate/kg BW. It was found that carbohydrate source and level of cotton seed meal did not have significant effects on ruminal pH, ammonia nitrogen concentration, microbial protein synthesis or feed intake. Animals which received CC showed significantly higher BUN concentration, ruminal propionic acid and butyric acid proportions, while dry matter, organic matter digestibility, populations of total viable bacteria and proteolytic bacteria were lower than those in the CR3:1 treatment. The concentration of total volatile fatty acids was higher in HCM than LCM treatments, while the concentration of butyric acid was higher in LCM than HCM treatments. The population of proteolytic bacteria with the LCM treatments was higher than the HCM treatments; however other bacteria groups were similar among the different levels of cotton seed meal. Bulls which received LCM had higher protein digestibility than those receiving HCM. Therefore, using high levels of cassava chip and cotton seed meal might positively impact on energy and nitrogen balance for the microbial population in the rumen of the young dairy bull.

Detection for Methicillin Resistant Staphylococcus aureus in Using Bio-Chip Based Loop Mediated Isothermal Amplification Assay (칩 기반 등온증폭법을 이용한 약제 내성 포도상구균의 검출)

  • Cho, Min-Ho;Jang, Won-Cheoul;Choi, Jae-Gu
    • Journal of the Korean Chemical Society
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    • v.57 no.1
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    • pp.81-87
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    • 2013
  • Staphylococcus aureus is the most important pathogen in nosocomial infections, including bloodstream infections. Prompt identification of S. aureus from blood cultures and detection of methicillin resistance are essential in cases of suspected sepsis. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using intercalating reaction by addition of SYBR Green to amplicons of LAMP, and it's a unique gene amplification method in which DNA can be isothermally amplified using only one enzyme. Staphylococcus-LAMP, which targets the spa gene, encoding S.aureus-specific protein A, and the mecA gene, encoding penicillin-binding protein-2' for methicillin resistance, detected MRSA and MRSE. In this study, by using LAMP assay, I detected for Staphylococcus aureus and Staphylococcus epidermidis concentration in the clinical sample. The detection of Staphylococcus aureus and Staphylococcus epidermidis was tested by using serial 10-fold dilutions standard solution. I have accurate detected the limit of detection, sensitity, specificity and reproducibility of the assay. The Bio-chip based LAMP assay allowed easy, rapid, accurate and sensitive detection of infection with Staphylococcus and especially applicable in a resource-limited situation.

BEAD BASED CHEMICAL REACTION SYSTEM USING TEMPERATURE AND FLUID CONTROL FOR CANCER DETECTION (유체와 온도 조절을 이용한 생화학 물질 반응용 마이크로칩의 개발)

  • Kim, Min-Su;Lee, Bo-Rahm;Yoon, Hyo-Jin;Kim, Byung-Gee;Lee, Yoon-Sik;Kim, Yong-Kweon
    • Proceedings of the KIEE Conference
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    • 2008.07a
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    • pp.1466-1467
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    • 2008
  • We describe here a novel micro total analysis system for the purification and identification of the affinity-captured proteins. Also we demonstrated the mass analysis of the Carcinoembrionic antigen (CEA) and Alpha femtoprotein which were chosen as the target cancer marker. For MALDI-TOF analyses, the proteins should to be separated from a protein mixture and be concentrated when needed. This procedure usually takes a long time even before protease-digested samples are to be obtained from them. Here, we describe integrated and efficient micro chip for protein purification and digestion for MALDI-TOF analyses. At first, disease protein is purified by passing the micro chamber from a protein mixture or human whole serum and released from the micro affinity beads by thermal heating. Purified protein is then transfer to the hole for trypsin digestion. The final sample is analyzed by MALDI-TOF. All the processes could be finished successfully within one hour, which renders MALDI-TOF analyses of a target protein quite simple.

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Biochemical Application of IgG Fc-binding peptide: From Biochip to Targeted Nano Carrier

  • Chung, Sang Jeon
    • Proceedings of the Korean Vacuum Society Conference
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    • 2013.02a
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    • pp.84-84
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    • 2013
  • FcBP consisting of 13 amino acids specifically binds to Immunoglobulin G Fc domain. Initially, we utilized this peptide for preparation of antibody chip as a PEG composite for enhanced solubility. After then, the peptide conjugate was immobilized on agarose resin, resulting in highly efficient affinity column for antibody purification. The efficiency was comparable to commercial Protein A column. Recently, this peptide was conjugated with cell penetratingpeptide (CPP) on a backbone of GFP, affording antibody transducer, which carries antibody into live cells by simple mixing of antibody and the transducer in cell culture media. Antibody transduction into cells was monitored by live cell imaging. More recently, the FcBP was fused to ferritin cage, which consists of 24 ferritin protein molecules. The FcBP-ferritin cage showed greatly increased binding affinity to human IgG. Its binding was analyzed by QCM and SPR analysis. Finally, it was selectively delivered by Herceptin to SKBR3, a breast cancer cell, over MCF10A, non-tumorigenic cells.

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Dynamic MRM Measurements of Multi-Biomarker Proteins by Triple-Quadrupole Mass Spectrometry with Nanoflow HPLC-Microfluidics Chip

  • Ji, Eun-Sun;Cheon, Mi-Hee;Lee, Ju-Yeon;Yoo, Jong-Shin;Jung, Hyun-Jin;Kim, Jin-Young
    • Mass Spectrometry Letters
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    • v.1 no.1
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    • pp.21-24
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    • 2010
  • The development of clinical biomarkers involves discovery, verification, and validation. Recently, multiple reaction monitoring (MRM) coupled with stable isotope dilution mass spectrometry (IDMS) has shown considerable promise for the direct quantification of proteins in clinical samples. In particular, multiple biomarkers have been tracked in a single experiment using MRM-based MS approaches combined with liquid chromatography. We report here a highly reproducible, quantitative, and dynamic MRM system for validating multi-biomarker proteins using Nanoflow HPLC-Microfluidics Chip/Triple-Quadrupole MS. In this system, transitions were acquired only during the retention window of each eluting peptide. Transitions with the highest MRM-MS intensities for the five target peptides from colon cancer biomarker candidates were automatically selected using Optimizer software. Relative to the corresponding non-dynamic system, the dynamic MRM provided significantly improved coefficients of variation in experiments with large numbers of transitions. Linear responses were obtained with concentrations ranging from fmol to pmol for five target peptides.

Effects of Different Levels of Crushed Bamboo Chip on Performance and Carcass Characteristics in Holstein Steers (파쇄 대나무 급여수준이 젖소 거세우의 성장과 도체특성에 미치는 영향)

  • 안병홍;강춘성;추교문;조희웅
    • Journal of Animal Science and Technology
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    • v.48 no.3
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    • pp.401-414
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    • 2006
  • Twenty eight Holstein steers 12 months old and weighing about 300kg were andomly allotted into one of four groups being fed ammoniated rice straw(ARS) and substituted 30%, 40% and 50% crushed bamboo chip for ARS to determine the effects of different levels of bamboo chip on performance, digestibility and carcass characteristics. Daily weight gain was reduced as the substitution levels of bamboo chip for ARS as a roughage source increased but there were no differences in daily weight gain between steers fed ARS alone and 30% bamboo chip for ARS. Concentrates intakes were not different between treatments by the substitution levels of bamboo chip for the whole fattening period. Roughage intake tended to increase as the substitution levels of bamboo chip increased. Total feed intake was not affected by the substitution levels of bamboo chip. However, feed efficiency got worse with increasing levels of bamboo chip. Animals fed the roughage substituting 30% bamboo chip for ARS were higher in profit by 13% than animals fed ARS alone as a roughage source. Digestibilities of Dry matter(DDM) and crude fiber(DCF) were highest in animals fed ARS alone as a roughage source. DDM's were lower in higher substitution levels of crushed bamboo chip but there were no differences in DCF among animals fed different levels of bamboo chip as a roughage source. Crude protein digestibility was not affected by ammoniated rice straw or by the different levels of bamboo chip. Dressing percentage and backfat thickness were not affected by ammoniated rice straw or by the levels of bamboo chip but ribeye area was narrowed as the levels of bamboo chip increased. Beef color, fat color, texture, maturity and marbling score were not affected by feeding of ammoniated rice straw or by the levels of bamboo chip. According to these results, it may be concluded that profit can increase when Holstein bulls are castrated and roughage containing ammoniated rice straw plus 30% bamboo chip is offered.