• Title/Summary/Keyword: Protein determination

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Novel Purification Method of Kv 4.2 Potassium Channel from Rat Brain Membrane

  • Park, Sung-Soo
    • Biomedical Science Letters
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    • v.18 no.2
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    • pp.96-103
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    • 2012
  • Kv 4.2 ion channel protein has an ability to open at subthreshold membrane potentials and to recover quickly from inactivation. That is very important for neuronal signal transmission in vertebrate brain. In order to purify Kv 4.2 protein, the novel purification methods were experimented. The purification procedure utilized chromatography on DE-52 ion exchange column and affinity chromatography on a WGA-Sepharose 4B, and Kv 4.2 affinity column chromatography. It was found that 0.5% (wt./vol.) Triton X-100 detergent in lysis buffer worked well for Kv 4.2 protein solubilization from rat brain membrane. Protein quantitative determination was conducted by BCA method at 562 nm for each purification step to avoid determination interference of protein at 280 nm by detergent. The confirmation of Kv 4.2 existence and amount is performed using by SDS-PAGE/immunoblotting or 96-well dot blotting. The Kv 4.2 without interacting protein that contains carbohydrate, was purified from novel biochemical 3-steps purification method for further research.

A Turbidimetric Determination of Protein by Trichloroacetic Acid

  • Choi, Wahn-Soo;Chung, Kae-Jong;Chang, Man-Sik;Chun, Jae-Kwang;Lee, Hyang-Woo;Hong, Sung-Youl
    • Archives of Pharmacal Research
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    • v.16 no.1
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    • pp.57-61
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    • 1993
  • Based on the turbidimetric response of protein with 50% trichloroacetic acid (TCA), this study aims to introduce an assay method for protein in solution. The standard procedure consists of mixing equal volume of sample solution (standard or unknown) with 50%-TCA solution and measuring the absorbance at 450 nm after 20 min. The absorbances of the solutions were almost stable over 120 min at room temperature. This assy method is simple, reproducible, and tolerant to many interfering substances. It can detect less amount than $10\mu$g/ml of bovin serum albumin. The assay method has low protein-to-protein variability over wide range of molecular weight.

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Sex Determination of Cattle Meat by Polymerase Chain Reaction Amplification of the DEAD Box Protein (DDX3X/DDX3Y) Gene

  • Gokulakrishnan, P.;Kumar, R.R.;Sharma, B.D.;Mendiratta, S.K.;Sharma, D.
    • Asian-Australasian Journal of Animal Sciences
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    • v.25 no.5
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    • pp.733-737
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    • 2012
  • Determination of sex origin of cattle meat by fast and reliable molecular methods is an important measure to ensure correct allocation of export refunds particularly in European countries and also female cattle (cow) slaughter is legally banned in India because of religious beliefs. Based on the DEAD box protein gene located on the X and Y chromosomes, 2 pair of primers were designed and the system of PCR was optimized. Upon PCR amplification, male tissue showed 2 bands, while female tissue resulted in only one band. The accuracy and specificity of the primers was assessed using DNA template extracted from cattle meat of known sex. The protocol was subjected to a blind test and showed 100% concordance, proving its accuracy and reliability.

Determination of Monoclonal Antibodies Capable of Recognizing the Native Protein Using Surface Plasmon Resonance

  • Kim, Deok-Ryong
    • BMB Reports
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    • v.34 no.5
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    • pp.452-456
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    • 2001
  • Surface plasmon resonance has been used for a biospecific interaction analysis between two macromolecules in real time. Determination of an antibody that is capable of specifically interacting with the native form of antigen is very useful for many biological and medical applications. Twenty monoclonal antibodies against the $\alpha$ subunit of E. coli DNA polymerase III were screened for specifically recognizing the native form of protein using surface plasmon resonance. Only four monoclonal antibodies among them specifically recognized the native $\alpha$ protein, although all of the antibodies were able to specifically interact with the denatured $\alpha$ subunit. These antibodies failed to interfere with the interaction between the $\tau$ and $\alpha$ subunits that were required for dimerization of the two polymerases at the DNA replication fork. This real-time analysis using surface plasmon resonance provides an easy method to screen antibodies that are capable of binding to the native form of the antigen molecule and determine the biological interaction between the two molecules.

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Preparation and Determination of Structure of L-3-Deoxymimosine-containing Peptides

  • Chae, Whi-Gun;Lee, Eung-Seok
    • Archives of Pharmacal Research
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    • v.23 no.3
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    • pp.211-221
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    • 2000
  • L-3-Deoxymimosine-containing decapeptides were prepared for the development of protein tyrosine kinase (PTK) inhibitors. During the preparation of peptides, several side products were formed. identification and determination of major peptides generated were reported.

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Application of Near-Infrared Reflectance Spectroscopy (NIR) Method to Rapid Determination of Seed Protein in Coarse Cereal Germplasm

  • Lee, Young-Yi;Kim, Jung-Bong;Lee, Ho-Sun;Lee, Sok-Young;Gwag, Jae-Gyun;Ko, Ho-Cheol;Huh, Yun-Chan;Hyun, Do-Yoon;Kim, Chung-Kon
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.55 no.4
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    • pp.357-364
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    • 2010
  • Kjeldahl method used in many materials from various plant parts to determine protein contents, is laborious and time-consuming and utilizes hazardous chemicals. Near-infrared (NIR) reflectance spectroscopy, a rapid and environmentally benign technique, was investigated as a potential method for the prediction of protein content. Near-infrared reflectance spectra(1100-2400 nm) of coarse cereal grains(n=100 for each germplasm) were obtained using a dispersive spectrometer as both of grain itself and flour ground, and total protein contents determined according to Kjeldahl method. Using multivariate analysis, a modified partial least-squares model was developed for prediction of protein contents. The model had a multiple coefficient of determination of 0.99, 0.99, 0.99, 0.96 and 0.99 for foxtail millet, sorghum, millet, adzuki bean and mung bean germplasm, respectively. The model was tested with independent validation samples (n=10 for each germplasm). All samples were predicted with the coefficient of determination of 0.99, 0.99, 0.99, 0.91 and 0.99 for foxtail millet, sorghum, millet, adzuki bean and mung bean germplasm, respectively. The results indicate that NIR reflectance spectroscopy is an accurate and efficient tool for determining protein content of diverse coarse cereal germplasm for nutrition labeling of nutritional value. On the other hands appropriate condition of cereal material to predict protein using NIR was flour condition of grains.

Determination of Protein and Oil Contents in Soybean Seed by Near Infrared Reflectance Spectroscopy

  • Choung, Myoung-Gun;Baek, In-Youl;Kang, Sung-Taeg;Han, Won-Young;Shin, Doo-Chull;Moon, Huhn-Pal;Kang, Kwang-Hee
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.46 no.2
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    • pp.106-111
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    • 2001
  • The applicability of near infrared reflectance spectroscopy(NIRS) was tested to determine the protein and oil contents in ground soybean [Glycine max (L.) Merr.] seeds. A total of 189 soybean calibration samples and 103 validation samples were used for NIRS equation development and validation, respectively. In the NIRS equation of protein, the most accurate equation was obtained at 2, 8, 6, 1(2nd derivative, 8 nm gap, 6 points smoothing and 1 point second smoothing) math treatment condition with SNV-D (Standard Normal Variate and Detrend) scatter correction method and entire spectrum by using MPLS (Modified Partial Least Squares) regression. In the case of oil, the best equation was obtained at 1, 4, 4, 1 condition with SNV-D scatter correction method and near infrared (1100-2500nm) region by using MPLS regression. Validation of these NIRS equations showed very low bias (protein:-0.016%, oil : -0.011 %) and standard error of prediction (SEP, protein: 0.437%, oil: 0.377%) and very high coefficient of determination ($R^2$, protein: 0.985, oil : 0.965). Therefore, these NIRS equation seems reliable for determining the protein and oil content, and NIRS method could be used as a mass screening method of soybean seed.

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Computational Approaches for Structural and Functional Genomics

  • Brenner, Steven-E.
    • Proceedings of the Korean Society for Bioinformatics Conference
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    • 2000.11a
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    • pp.17-20
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    • 2000
  • Structural genomics aims to provide a good experimental structure or computational model of every tractable protein in a complete genome. Underlying this goal is the immense value of protein structure, especially in permitting recognition of distant evolutionary relationships for proteins whose sequence analysis has failed to find any significant homolog. A considerable fraction of the genes in all sequenced genomes have no known function, and structure determination provides a direct means of revealing homology that may be used to infer their putative molecular function. The solved structures will be similarly useful for elucidating the biochemical or biophysical role of proteins that have been previously ascribed only phenotypic functions. More generally, knowledge of an increasingly complete repertoire of protein structures will aid structure prediction methods, improve understanding of protein structure, and ultimately lend insight into molecular interactions and pathways. We use computational methods to select families whose structures cannot be predicted and which are likely to be amenable to experimental characterization. Methods to be employed included modern sequence analysis and clustering algorithms. A critical component is consultation of the presage database for structural genomics, which records the community's experimental work underway and computational predictions. The protein families are ranked according to several criteria including taxonomic diversity and known functional information. Individual proteins, often homologs from hyperthermophiles, are selected from these families as targets for structure determination. The solved structures are examined for structural similarity to other proteins of known structure. Homologous proteins in sequence databases are computationally modeled, to provide a resource of protein structure models complementing the experimentally solved protein structures.

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Determination of Protein Content in Pea by Near Infrared Spectroscopy

  • Lee, Jin-Hwan;Choung, Myoung-Gun
    • Food Science and Biotechnology
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    • v.18 no.1
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    • pp.60-65
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    • 2009
  • Near infrared reflectance spectroscopy (NIRS) was used as a rapid and non-destructive method to determine the protein content in intact and ground seeds of pea (Pisum sativum L.) germplasms grown in Korea. A total of 115 samples were scanned in the reflectance mode of a scanning monochromator at intact seed and flour condition, and the reference values for the protein content was measured by auto-Kjeldahl system. In the developed ground and intact NIRS equations for analysis of protein, the most accurate equation were obtained at 2, 8, 6, 1 math treatment conditions with standard normal variate and detrend scatter correction method and entire spectrum (400-2,500 nm) by using modified partial least squares regression (n=78). External validation (n=34) of these NIRS equations showed significant correlation between reference values and NIRS estimated values based on the standard error of prediction (SEP), $R^2$, and the ratio of standard deviation of reference data to SEP. Therefore, these ground and intact NIRS equations can be applicable and reliable for determination of protein content in pea seeds, and non-destructive NIRS method could be used as a mass analysis technique for selection of high protein pea in breeding program and for quality control in food industry.