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Analytical Biochemistry

Brandon L Doyle, Ivan L Budyak, Adam P Rauk, William F Weiss
Appropriate characterization of soluble aggregates is an important aspect of biologics development and manufacturing, and sedimentation velocity analytical ultracentrifugation (SV-AUC) is often used an orthogonal technique to size-exclusion chromatography (SEC) for this purpose. Precise quantification of low levels of soluble aggregates by SV-AUC can be adversely impacted by improper cell alignment. This report describes the development of an optical system capable of quantifying cell alignment that affords a substantial improvement compared to historical approaches...
May 18, 2017: Analytical Biochemistry
Liangfu Zhu, Ramachandram Badugu, Douguo Zhang, Ruxue Wang, Emiliano Descrovi, Joseph R Lakowicz
Fluorescence spectroscopy and imaging are now used throughout the biosciences. Fluorescence microscopes, spectrofluorometers, microwell plate readers and microarray imagers all use multiple optical components to collect, redirect and focus the emission onto single point or array imaging detectors. For almost all biological samples, except those with regular nanoscale features, emission occurs in all directions. With the exception of complex microscope objectives with large collection angles (NA ≤ 0.5), all these instruments collect only a small fraction of the total emission...
May 17, 2017: Analytical Biochemistry
Hedi Sinijarv, Shanshan Wu, Taavi Ivan, Kaido Viht, Asko Uri
High demand for inhibitors regulating the activity of protein kinases has stimulated the quest for high throughput and reliable compound screening assays. Here we introduce a method applying a non-metal photoluminescent probe ARC-Lum(Fluo) for determination of dissociation constants of competitive inhibitors of protein kinases. Employing a single probe instead of a combination of antibody and fluorescent tracer makes the assay simpler, cheaper, and more accurate than several other inhibitor-screening technologies...
May 17, 2017: Analytical Biochemistry
Sa Dong, Cunzheng Zhang, Yuan Liu, Xiao Zhang, Yajing Xie, Jianfeng Zhong, Chongxin Xu, Xianjin Liu
The detections of Cry1 toxins are mainly dependent on immunoassays based on specific monoclonal antibodies (mAb). In the present study, a mixture immunization with seven Cry1 toxins was administered. The results showed that five mAbs with different characteristics, especially one mAb named 5-E8 which could recognize all the seven Cry1 toxins were obtained. Based on the 5-E8 mAb, a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) which can specifically detect the seven Cry1 toxins without cross-reactivity to Cry2A and vip3 was developed with the limit of detection (LOD) and limit of quantification (LOQ) of 6...
May 17, 2017: Analytical Biochemistry
Xiaolan Yang, Yiran Feng, Huimin Chong, Deqiang Wang, Xiaolei Hu, Jun Pu, Chang-Guo Zhan, Fei Liao
High-throughput estimation of specific activities of an enzyme and its mutants in a group (enzyme/mutants) in cell lysates via high-throughput assay of their activities and separate immunoturbidimetric assay (ITA) of their proteins was proposed. Pseudomonas aeruginosa arylsulfatase (PAAS) and Bacillus fastidious uricase (BFU) served as two models. ITA employed 0.75 mg of antisera against PAAS or BFU as the reference in 96-well microplates to measure the difference of extinction at 340 and 700 nm. According to the calibration curve, ITA quantified the reference from 0...
May 16, 2017: Analytical Biochemistry
Cristina Cudalbu, Arthur J L Cooper
No abstract text is available yet for this article.
May 15, 2017: Analytical Biochemistry
A-Young Lee, Na-Reum Ha, In-Pil Jung, Sang-Heon Kim, A-Ru Kim, Moon-Young Yoon
Antibiotics are useful for improving the living conditions of livestock. However, residual antibiotics induce several human diseases such as food-borne illness and infection of carbapenem-resistant Enterobacteriaceae (CRE). In this study, the identification of a benzylpenicillin-specific aptamer was selected by rGO-SELEX (reduced Graphene Oxide-Systematic Evolution of Ligands by EXponential enrichment). A random ssDNA library was incubated with rGO for adsorption and eluted with benzylpenicillin. As a result of the selection process, a DNA aptamer was found that specifically bound to benzylpenicillin with high binding affinity, Kd = 383...
May 15, 2017: Analytical Biochemistry
Satoru Ishihara, Naoe Kotomura, Naoki Yamamoto, Hiroshi Ochiai
Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence...
May 11, 2017: Analytical Biochemistry
Samanta Hernández-García, María Inmaculada García-García, Francisco García-Carmona
An improved method based on the p-nitrophenyl long chain esters method is proposed for measuring lipase hydrolytic activity in aqueous media. Using ethylene glycol as co-solvent for hydrophobic p-nitrophenyl substrates in aqueous buffer, lipase activity is measured by following the release of p-nitrophenol. This fast and easy to handle method improves the solubility of both substrate and product, and also the stability of the substrate. It avoids the use of solvents such as ethanol or propanol, permits the comparison of all the p-nitrophenol acyl ester substrates and allows the influence of ions like Ca(+2) to be studied, while avoiding turbidity in the reaction medium...
May 11, 2017: Analytical Biochemistry
Franziska Steinicke, Imke Oltmann-Norden, Hermann Wätzig
This work presents an extensive parameter list that facilitates a survey of biosensor performance using Biacore instruments for kinetic binding studies. Six long term measurements were performed using a strongly interacting antigen-antibody (β2 microglobulin) system. Both Single Cycle Kinetic (SCK) and Multi Cycle Kinetic (MCK) were executed each with five different analyte concentrations. The overall comparison of the long term monitored parameters, like the dissociation constant (KD with approximately 3-6% relative percental standard deviation), the association and dissociation rate constants (ka, kd), the analyte binding capacity (Rmax), chi(2) and the sum of the absolute values of the residuals, revealed the delicate factors that make the system performance vulnerable...
May 10, 2017: Analytical Biochemistry
Vasso Skouridou, Thomas Schubert, Abdulaziz S Bashammakh, Mohammad S El-Shahawi, Abdulrahman O Alyoubi, Ciara K O'Sullivan
In this work we report the mapping of the binding site of the only progesterone aptamer published to date, in an approach referred to as aptatope mapping. By linking the binding data obtained from microscale thermophoresis analysis to the structural differences on the ring structure of a range of steroids, we elucidated the moieties involved in aptamer-progesterone binding. This approach can be further exploited for the characterization of aptamer specificity and ultimately facilitate the development of aptamer-based assays depending on the desired specificity...
May 10, 2017: Analytical Biochemistry
Ge Chen, Maojun Jin, Pengfei Du, Chan Zhang, Xueyan Cui, Yudan Zhang, Yongxin She, Hua Shao, Fen Jin, Shanshan Wang, Lufei Zheng, Jing Wang
The chemiluminescence enzyme immunoassay (CLEIA) method responds differently to various sample matrices because of the matrix effect. In this work, the CLEIA method was coupled with molecularly imprinted polymers (MIPs) synthesized by precipitation polymerization to study the matrix effect. The sample recoveries ranged from 72.62% to 121.89%, with a relative standard deviation (RSD) of 3.74-18.14%.The ratio of the sample matrix-matched standard curve slope rate to the solvent standard curve slope was 1.21, 1...
May 9, 2017: Analytical Biochemistry
Melissa J Blacketer, Margaret K Gannon, Isaac A Young, Michael A Shogren-Knaak
DNA templates for assembling chromatin model systems typically consist of numerous repeats of nucleosome positioning sequences, making their synthesis challenging. Here we describe a solid-phase strategy for generating such templates using sequential enzymatic ligation of DNA monomers. Using single nucleosome site monomers, we can either generate a twelve-nucleosome site target, or systematically access intermediate-sized templates. Using twelve nucleosome positioning site monomers, longer templates can be generated...
May 8, 2017: Analytical Biochemistry
Meng-Xi Li, Xing-Hua Wang, Lian-Ming Zhang, Xiao-Ping Wei
To improve the sensitivity of the molecular imprinting sensor detection of protein, a new strategy based on enzyme amplification was proposed. The determination of bovine serum albumin (BSA) was achieved by using the epitope imprinted techniques coupling with electrochemical measurement method. Nonapeptide, separated from BSA, was selected as a template molecule to prepare the molecularly imprinted polymer (MIP) film, and it could bind with the cavities of the MIP. By the use of epitope imprinted techniques, BSA can be recognized by the MIP via the nonapeptide on the surface of BSA...
May 5, 2017: Analytical Biochemistry
Le Cong
Years of advances in high-throughput biotechnologies exemplified by nucleic acid sequencing and single-molecular imaging have led to our increasing capacity to interrogate genomes down to nucleotide accuracy with single-cell or even subcellular resolution, thereby gaining high-dimensional information on the genetic variants and epigenetic states associated with physiological and pathological processes. To achieve a causal understanding the exquisite biology encoded in our genome, researchers in the past decades have sought to develop companion genome engineering tools...
May 4, 2017: Analytical Biochemistry
Sung-Hee Oh, Yong-Bok Choi, June-Hyun Kim, Conrad C Weihl, Jeong-Sun Ju
Macroautophagy (hereafter referred to as autophagy) is a degradation system that delivers cytoplasmic materials to lysosomes via autophagosomes. Autophagic flux is defined as a measure of autophagic degradation activity. Despite several methods for monitoring autophagic flux being currently utilized, interest in finding a highly accurate, sensitive and well-quantifiable assay is still growing. Therefore, we introduce a new approach analyzing autophagic flux in vitro and in vivo using enzyme-linked immunosorbent assay (ELISA) technique...
May 4, 2017: Analytical Biochemistry
Kamlesh Bisht, Sherilyn Grill, Jacqueline Graniel, Jayakrishnan Nandakumar
CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells...
May 4, 2017: Analytical Biochemistry
Marie Reille-Seroussi, Jean-François Gaucher, Laure-Anne Cussac, Isabelle Broutin, Michel Vidal, Sylvain Broussy
The VEGFR1 has been shown to play a role in the regulation of angiogenesis, and has therefore been associated to several pathologies. In order to extend our toolbox of screening methods for the identification of compounds disrupting the VEGF receptor 1/VEGF interaction, we developed a fast and accurate displacement assay, in which VEGF receptor 1 domain 2 is directly labeled with an enzyme, bypassing the classical streptavidin-biotin interaction system. A description of this straightforward strategy is provided here, including its advantages and disadvantages...
May 3, 2017: Analytical Biochemistry
Simon Godin, Diego Bouzas-Ramos, Stéphanie Fontagné-Dicharry, Brice Bouyssière, Maïté Bueno
Studies have shown that information related to the presence of low-molecular-weight metabolites is frequently lost after deproteinization of complex matrices, such as blood and plasma, during sample preparation. Therefore, the effect of several deproteinization reagents on low-molecular-weight selenium species has been compared by species-specific isotope labeling. Two isotopically enriched selenium tracers were used to mimic models of small inorganic anionic ((77)Se-selenite) and organic zwitterionic ((76)Se-selenomethionine) species...
May 3, 2017: Analytical Biochemistry
Imeobong U Antia, Darshna R Yagnik, Leonardo Pantoja Munoz, Ajit J Shah, Frank A Hills
Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 μg GAG...
April 30, 2017: Analytical Biochemistry
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