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Analytical Biochemistry

Seyed Hamid Jalalian, Mohammad Ramezani, Seyed Ali Jalalian, Khalil Abnous, Seyed Mohammad Taghdisi
Exosomes are endosomal-derived vesicles, playing a major role in cell-to-cell communication. Multiple cells secret these vesicles to induce and inhibit different cellular and molecular pathways. Cancer-derived exosomes have been shown to affect development of cancer in different stages and contribute to the recruitment and reprogramming of both proximal and distal tissues. The growing interest in defining the clinical relevance of these nano-sized particles in cancers, has led to the identification of either tissue- or disease-specific exosomal contents, such as nucleic acids, proteins and lipids as a source of new biomarkers which propose the diagnostic potentials of exosomes in early detection of cancers...
February 15, 2019: Analytical Biochemistry
Yuta Yamamoto, Tetsuya Saita, Rintaro Sogawa, Kenji Ogata, Yutaro Yamamoto, Sakiko Kimura, Yutaka Narisawa, Shinya Kimura, Masashi Shin
The tyrosine kinase inhibitor ponatinib is extensively metabolized in the body, and consequently the development of specific immunoassays for pharmacokinetic studies and therapeutic drug monitoring of ponatinib is challenging. If two antibodies simultaneously recognize the entire structure of ponatinib, they could be utilized to establish an ultra-specific sandwich immunoassay for ponatinib, free of any interference from ponatinib metabolites. In this study, we created two types of anti-ponatinib polyclonal antibodies that recognize two different ponatinib epitopes, and sandwiched almost all structural components of ponatinib in these two antibodies in order to develop an enzyme-linked immunosorbent assay (ELISA) technique not affected by any ponatinib metabolites...
February 13, 2019: Analytical Biochemistry
Barbara Bobrowska-Korczak, Paulina Gątarek, Angelina Rosiak, Joanna Giebultowicz, Joanna Kałużna-Czaplińska
The aim of this study was to investigate and compare the levels of concentration of modified nucleosides in the urine of autistic and healthy children. The compounds have never been analyzed before. The levels of nucleosides in the urine of both groups were determined by validated high performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. Chromatographic separation was achieved with HILIC column and tubercidin was used as the internal standard for the quantification of urinary nucleosides...
February 13, 2019: Analytical Biochemistry
So-Young Lee, Jae-Cheong Lim, Eun-Ha Cho, Seung-Kon Lee, Sung-Hee Jung
Scintillation proximity assay (SPA) is a type of radioimmunoassay (RIA). We apply ultrasound enhancement to the general SPA. All assay procedures, including the antibody coating and radiolabeled antigen binding are achieved by simply mixing then standing for 5 min in an ultrasound chamber. No additional incubation time is required. To further demonstrate the capability of the UE-SPA, a quantitative measurement of CD55 in various grades of colon tumors was assessed on human tissue slides. The results showed a significant correlation between CD55 expression and tumorigenesis...
February 13, 2019: Analytical Biochemistry
J E Abud, C G Santamaría, M Oggero, H A Rodriguez
One of the most used formats in inmuno-polymerase chain reaction (IPCR) is known as "Universal" IPCR (signal-generating complexes is based on conjugates of biotinylated DNA, biotinylated IgG and avidin). In the present study, we evaluated the utility of using mono- and bi-biotinylated DNA probes, pre-self-assembled DNA-neutravidin complex, blocking step and glutaraldehyde pretreatment of standard PCR tubes to improve the analytical performance of the hTSH-IPCR assay. The use of pre-self-assembled mono-biotinylated DNA-neutravidin complex enhances both the sensitivity and the reproducibility of the hTSH-IPCR assay, even without blocking step: hTSH-IPCR assay showed an improved limit of detection (LOD: 0...
February 12, 2019: Analytical Biochemistry
Anna Davydova, Mariya Vorobyeva, Eugenia Bashmakova, Pavel Vorobjev, Olga Krasheninina, Alexey Tupikin, Marsel Kabilov, Vasilisa Krasitskaya, Ludmila Frank, Alya Venyaminova
Aptamers are short DNA and RNA fragments which bind their molecular targets with affinity and specificity comparable to those of antibodies. Here, we describe the selection of novel 2'-F-RNA aptamers against total human hemoglobin or its glycated form HbA1c. After SELEX and high-throughput sequencing of the enriched libraries, affinities and specificities of candidate aptamers and their truncated variants were examined by the solid-phase bioluminescent assay. As a result, we identified aptamers specific to both hemoglobins or only glycated HbA1c...
February 8, 2019: Analytical Biochemistry
Conny Unger, Nicole Lokmer, Daniel Lehmann, Ilka M Axmann
Residual phenol, carried over from RNA purification, can alter RNA concentration measurements and is assumed to inhibit PCR. Here, we demonstrate that Impurities A260 values of spectral content profiling (SCP) UV/Vis measurements correlated with phenol concentration, whereas absorbance ratios of classical UV/Vis systems failed to reliably detect phenol in RNA samples. Phenol contamination led to over- or underestimation of RNA concentration on UV/Vis systems, whereas it had no influence on fluorometry quantification...
February 8, 2019: Analytical Biochemistry
Shigeru Ueda, Shin-Ichi Sakasegawa
Previously, we developed a kinase cycling method using creatine kinase and pyruvate kinase (RMPK) both from rabbit muscle in the presence of an excess amount of ATP and IDP for the quantitative determination of substrate. To our surprise, the RMPK cycling reaction was 10-fold more efficient using Mn2+ rather than Mg2+ . Here, we investigated PK from Geobacillus stearothermophilus (GSPK) as an alternative source of enzyme. Spectrophotometric real-time detection was accomplished by coupling the reaction to ADP-dependent glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase (G6PD)...
February 7, 2019: Analytical Biochemistry
Himanshu Singh, Deepshikha Verma, Benjamin Bardiaux
We report the observation of single-site phosphorylation in a His-tag sequence N-terminally attached to a recombinant protein (UVI31+) in vitro. This modification was detected at position 23 at a serine residue of the His-tag sequence encoded by the vector pET28a. Furthermore, the phosphorylated tag sequence was found to be dephosphorylated by the action of alkaline phosphatases. The functional activity and dynamics of the protein carrying the His-tag sequence were unchanged after phosphorylation. The possibility of phosphorylation within the N-terminal His-tag demonstrates that care has to be taken upon analysis of post-translational modifications via mass spectrometry for recombinant protein expression strategies...
February 5, 2019: Analytical Biochemistry
Janne Kulpakko, Kaisu Rantakokko-Jalava, Erkki Eerola, Pekka E Hänninen
Urinary tract infections (UTIs) are a common problem worldwide. The most prevalent causative pathogen of UTI is Escherichia coli, focus of this study. The current golden standard for detecting UTI is bacterial culture, creating a major workload for hospital laboratories - cost-effective and rapid mass screening of patient samples is needed. Here we present an alternative approach to screen patient samples with a single-step assay utilising time-resolved luminescence and luminescence modulating biosensing phages...
February 5, 2019: Analytical Biochemistry
Xiaoying Wang, Patrycja Magdziarz, Ernest Enriquez, Wang Zhao, Chris Quan, Narek Darabedian, Jamil Momand, Feimeng Zhou
Docking on the p53-binding site of murine double minute 2 (MDM2) by small molecules restores p53's tumor-suppressor function. We previously assessed 3244 FDA-approved drugs via "computational conformer selection" for inhibiting MDM2 and p53 interaction. Here, we developed a surface plasmon resonance method to experimentally confirm the inhibitory effects of the known MDM2 inhibitor, nutlin-3a, and two drug candidates predicted by our computational method. This p53/MDM2 interaction displayed a dosage-dependent weakening when MDM2 is pre-mixed with drug candidates...
February 2, 2019: Analytical Biochemistry
Matthias Wurm, Sibel Ilhan, Uwe Jandt, An-Ping Zeng
Utilizing flow cytometry to monitor progress of bulk biochemical reactions and concentration of chemical species normally relies on the utilization of cells carrying intrinsic fluorescence or modified beads. We present a method for a simple measurement of the fluorescent marker molecule fluorescein and GFPuv in bulk solutions with high sensitivity using a CytoFLEX flow cytometer and without the need for modified beads. Polystyrene beads were used to trigger measurements based on their high scatter signal, to detect the fluorescence signal from two different fluorophores present in the sample solution...
January 30, 2019: Analytical Biochemistry
Mona Al-Mugotir, Carol Kolar, Krysten Vance, David L Kelly, Amarnath Natarajan, Gloria E O Borgstahl
Due to the therapeutic potential of targeting protein-protein interactions (PPIs) there is a need for easily executed assays to perform high throughput screening (HTS) of inhibitors. We have developed and optimized an innovative and robust fluorescence-based assay for detecting PPI inhibitors, called FluorIA (Fluorescence-based protein-protein Interaction Assay). Targeting the PPI of RAD52 with replication protein A (RPA) was used as an example, and the FluorIA protocol design, optimization and successful application to HTS of large chemical libraries are described...
January 29, 2019: Analytical Biochemistry
Yeoseon Kim, Dabin Lee, Jeongkwon Kim
The effects of incubation temperature and acetonitrile (ACN) amount on microwave-assisted tryptic digestion of horse skeletal muscle myoglobin (MYG) and bovine serum albumin (BSA) were investigated. Microwave-assisted tryptic digestion was performed on BSA or MYG solutions containing different amounts (0, 10, and 20%) of ACN for different times (10, 20, 30, 40, and 50 min) at different temperatures (25, 37, and 55 °C). Conventional overnight tryptic digestion was also conducted with gentle mixing at 37 °C for 16 h...
January 29, 2019: Analytical Biochemistry
Mohtaram Ramezanpour, Shahram Naghizadeh Raeisi, Seyed-Ahmad Shahidi, Sorour Ramezanpour, Shahram Seidi
In this work, a novel sorbent based on polydopamine-functionalized magnetic ferric oxide (Fe3 O4 ) was synthesized and applied for dispersive solid phase extraction of Pb(II) in bovine milk samples. The extracts were analyzed by flame atomic absorption spectrometry (FAAS). The sorbent was characterized with different analytical techniques (XRD, FT-IR, SEM, TEM, VSM and EDX). To reach the maximum extraction efficiency of Pb(II), some effective parameters on both adsorption and desorption steps were optimized with the aid of central composite design and response surface methodology...
January 25, 2019: Analytical Biochemistry
Deshui Yu, Ju Zhang, Guangxuan Tan, Ningshu Yu, Qiuyue Wang, Qiqi Duan, Xin Qi, Mingjiao Cheng, Chunxue Yan, Zhangkun Wei, Zhenmiao Yu, Wenchao Huang, Chengwei Li
Genomic DNA isolation is a crucial technique for researchers studying plant molecular biology. A current widely-used protocol for DNA extraction needs a pestle and mortal for each sample and consumes a large amount of liquid nitrogen in grinding the samples. Most high-throughput methods depend on expensive machines for tissue homogenization. Here we developed a CTAB-based DNA extraction method using 2.0 ml microcentrifuge tubes for sample processing. This protocol has the advantages that it is suitable for a variety of plants, easily-performed without special equipment, and high-throughput; it effectively avoids sample cross-contamination, and is inexpensive, rapid and safe...
January 24, 2019: Analytical Biochemistry
Ahsan Ahmad, Swakkhar Shatabda
RNA editing process like Adenosine to Intosine (A-to-I) often influences basic functions like splicing stability and most importantly the translation. Thus knowledge about editing sites is of great importance in molecular biology. With the growth of known editing sites, machine learning or data centric approaches are now being applied to solve this problem of prediction of RNA editing sites. In this paper, we propose EPAI-NC, a novel method for prediction of RNA editing sites. We have used l-mer composition and n-gapped l-mer composition as features and used Pearson Correlation Coefficient to select features according to Pareto Principle...
January 18, 2019: Analytical Biochemistry
M Laštovičková, P Bobál, D Strouhalová, J Bobálová
The main objective of this study was to develop an effective in-gel trypsin digestion protocol using aqueous-acetonitrile solvent system to facilitate MS analysis and maximize the number of identified proteins from biological samples. The procedure, where 80% acetonitrile was present in the trypsin reaction mixture, increased the number of matched peptides, and allowed the identification of more proteins with higher coverage than the common digestion protocol. Vimentin, annexins, tubulin, actin, peptidyl-prolyl cis-trans isomerase or alpha-enolase are examples of important proteins that change during the progress cancer...
January 17, 2019: Analytical Biochemistry
Kim N Ingenbosch, Anna Rousek, Dennis Wunschik, Kerstin Hoffmann-Jacobsen
A new method for the analysis of lipase activity in the immobilized state is developed. The fluorescence assay aims to quantify the potential of lipases for the application in organic solvents. As lipases are universally immobilized on polymeric carriers for the use in bioorganic synthesis, the assay includes an immobilization step on the walls of polymeric cuvettes. The activity of the immobilized lipase is probed by 4-methylumbelliferone hydrolysis. The activity retention as a function of solvent concentration is used as a measure for the solvent resistance of the enzyme variant...
January 17, 2019: Analytical Biochemistry
Yunyu Yi, Li Zang
Monoclonal antibody (mAb), one of the major types of therapeutic proteins in the pharmaceutical industry, is predominantly manufactured using mammalian cell culture [1]. Oxidative stress, potentially present during cell culture process, may increase the protein carbonyl content in the mAb product, which was reported to positively correlate with aggregate burst rate during storage [2]. In order to monitor carbonyl content during mAb process development, we developed a high-throughput screening method for therapeutic mAbs using size-exclusion chromatography followed by ultraviolet and fluorescence detection (SEC-UV/FL), optimized from a fluorescein thiosemicarbazide (FTC) semi-microplate method...
January 14, 2019: Analytical Biochemistry
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