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Analytical Biochemistry

Na Ying, Taifan Sun, Zhibao Chen, Guangping Song, Bingyao Qi, Shengjun Bu, Xiuwei Sun, Jiayu Wan, Zehong Li
MicroRNAs (miRNAs) have key roles in gene expression and can be employed as biomarkers for early diagnosis of various diseases, especially cancers. Detection of miRNAs remains challenging and often requires detection platforms. Here, a horseradish peroxidase (HRP)-assisted hybridization chain reaction (HCR) for colorimetric detection of miR-155 was described. In the presence of target miRNA, the capture probe immobilized on the microplate sandwiched the target miR-155 with the 3' end of the reporter probe. Another exposed part of the RP at the 5'end triggered HCR producing double-stranded DNA polymers with multiple fluorescein isothiocyanates (FITC) for signal amplification...
April 20, 2017: Analytical Biochemistry
Kenichiro Furuki, Toshimasa Toyo'oka
An in-line size-exclusion (SE) ultra-high-performance liquid chromatography (UHPLC)- 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) method to quantify thiols in monoclonal antibodies (mAb) when manufacturing antibody-drug conjugates (ADCs) was developed. The mAbs are separated on an SE-UHPLC column and monitored with a UV detector at a wavelength of 280 nm. Eluents are channeled into a reaction coil and mixed with DTNB to form 5-thio-2-nitrobenzoic acid (TNB). Thiol concentration is calculated using absorption at 412 nm...
April 18, 2017: Analytical Biochemistry
Himanshu Joshi, Vikas Jain
Rapid and high-throughput protein purification methods are required to explore structure and function of several uncharacterized proteins. Isolation of recombinant protein expressed in Escherichia coli strain BL21 (DE3) depends largely on the efficient and speedy bacterial cell lysis, which is considered as the bottleneck during protein purification. Cells are usually lysed by either sonication or high pressure homogenization, both of which are slow, require special equipment, lead to heat generation, and may result in loss of protein's biological activity...
April 18, 2017: Analytical Biochemistry
Hui Li, Wei Shen, Michael Hon-Wah Lam, Haojun Liang
Intracellular delivery of foreign DNA probes sharply increases the efficiency of various biodetection protocols. Spherical nucleic acid (SNA) conjugate is a new type of probe that consists of a dense oligonucleotide shell attached typically to a gold nanoparticle core. They are widely used as novel labels for in vitro biodetection and intracellular assay. However, the degradation of foreign DNA still remains a challenge that can cause significant signal leakage (false positive signal). Hence, the site and behavior of intracellular degradation need to be investigated...
April 17, 2017: Analytical Biochemistry
M Fatih Canbolat, Hasan Basri Savas, Fatih Gultekin
This study examines the effects of CD use on enzymatic activity, following enzyme immobilization into nanofibers. There is almost no research available on the change in enzyme activity following interaction with cyclodextrin and electrospun nanofiber mats together. Laccase enzyme was immobilized into nanofibrous structures by various techniques, with and without γ-CD addition, and the enzymatic activity of the laccase was analyzed. SEM, XRD, and FTIR analyses were used for the characterization of the resulting structures...
April 15, 2017: Analytical Biochemistry
Saroj Kumar, Rajesh Ahirwar, Ishita Rehman, Pradip Nahar
Rapid diagnostic tests can be developed using ELISA for detection of diseases in emergency conditions. Conventional ELISA takes 1-2 days, making it unsuitable for rapid-diagnostics. Here, we report the effect of reagents mixing via shaking or vortexing on the assay timing of ELISA. A 48-min protocol of ELISA involving 12-min incubations with reagent mixing at 750 rpm of for every step was optimized. Contrary to this, time-optimized control ELISA performed without mixing produced similar results in 8 h, leaving a time gain of 7 h using the developed protocol...
April 14, 2017: Analytical Biochemistry
Anton G Rogov, Alexandra P Ovchenkova, Tatiana N Goleva, Igor I Kireev, Renata A Zvyagilskaya
The overwhelming majority of investigations on mitochondrial morphology were performed using S. cerevisiae. In this study we showed the benefits of applying new model organisms including petite-negative D. magnusii and Y. lipolytica yeasts for visualization of mitochondrial fragmentation. Normally giant D. magnusii cells and filament-like Y. lipolytica cells contain the highly structured mitochondrial reticulum. Oxidative stress mediated by tert-butyl hydroperoxide triggered mitochondrial fragmentation in yeasts...
April 12, 2017: Analytical Biochemistry
Dagmar Rissel, Peter Paul Heym, Edgar Peiter
Poly(ADP-ribose) polymerases (PARPs) have been implicated in responses of plants to DNA damage and numerous stresses, whereby the mechanistic basis of the interference is often unclear. Therefore, the identification of specific inhibitors and potential interactors of plant PARPs is desirable. For this purpose, we established an assay based on heterologous expression of PARP genes from the model plant Arabidopsis thaliana in yeast. Expression of AtPARPs caused an inhibition of yeast growth to different extent, which was alleviated by inhibitors targeted at human PARPs...
April 10, 2017: Analytical Biochemistry
John C Sutherland
A method for obtaining fluorescence polarization data from an instrument designed to measure circular and linear dichroism is compared with a previously reported approach. The new method places a polarizer between the sample and a detector mounted perpendicular to the direction of the incident beam and results in determination of the fluorescence polarization ratio, whereas the previous method does not use a polarizer and yields the fluorescence anisotropy. A similar analysis with the detector located axially with the excitation beam demonstrates that there is no frequency modulated signal due to fluorescence polarization in the absence of a polarizer...
April 6, 2017: Analytical Biochemistry
Lidia Eremina, Natalya Pashintseva, Leonid Kovalev, Marina Kovaleva, Sergey Shishkin
The mitochondrial set of proteins is a dynamic system, crucial for multiple functions of this organelle. Differential expression of genes in various tissues, alternative splicing, post-translational modifications, turnover and spatial dynamics of proteins are the factors that influence mitochondrial proteomes increasing their versatility. A wide range of high-throughput proteomic approaches are extensively used for identification, quantification and functional assessment of human and other mammalian mitochondrial proteins...
April 3, 2017: Analytical Biochemistry
Talia Miron, Meir Wilchek
A colorimetric method for determining cyanuric chloride (CC) and for monitoring its polysaccharide gel activation, before and after ligand binding, was developed. The method is based on the reaction of CC or its activated gel with pyridine and barbituric acid or dimethylbarbituric acid. The product formed yields a purple red color with λ max at 595 nm, and an EM value of approximately 300,000 after 1 h at room temperature. Due to its high sensitivity, this method can detect traces of CC (1 μM) under the above conditions...
April 1, 2017: Analytical Biochemistry
Bakri M Assas, Wesam H Abdulaal, Majed H Wakid, Haytham A Zakai, J Miyan, J L Pennock
Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation...
March 31, 2017: Analytical Biochemistry
Mohammad Bagher Gholivand, Elahe Ahmadi, Mozhdeh Haseli
In the present study, a graphite electrode (GE) modified by conductive film (containing functionalized multi-walled carbon nanotubes (f-MWCNTs), poly methylene blue p(MB) and gold nanoparticles (AuNPs)) was introduced for determination of nevirapine (NVP) as an anti-HIV drug by applying the differential pulse anodic stripping voltammetry (DPASV) technique. Modification of the electrode was investigated by scanning electron microscopy (SEM) and impedance electrochemical spectroscopy (EIS). All electrochemical effective parameters on detection of NVP were optimized and the oxidation peak current of drug was used for its monitoring...
March 31, 2017: Analytical Biochemistry
Hana Tawarahara, Isao Kuraoka, Shigenori Iwai
We previously developed a method to detect the cellular ability of nucleotide excision repair, which functions to remove UV-induced lesions in DNA, using a plasmid-type fluorescent probe. A drawback to the popular use of this method was that the oligonucleotide containing the (6-4) photoproduct, which was used as a primer in the plasmid preparation, must be synthesized chemically. In this study, we prepared the probe using a post-synthetically UV-irradiated oligonucleotide as the primer. Transfection of cells demonstrated that this probe detected the repair ability of the cells in the same manner as the original probe...
March 30, 2017: Analytical Biochemistry
Özlem Ipek
Radiofrequency (RF) coils are key components of magnetic resonance imaging (MRI) systems. The primary purpose of this review is to provide a basic theory of RF coil designs and their characterization by bench measurements, electromagnetic field simulations and MR measurements. With the continuing increase of magnetic field strength in MRI instruments, the RF wavelength in the subject under study becomes comparable to or smaller in size than the anatomical dimensions of the tissue under study, which amplifies the signal inhomogeneity...
March 29, 2017: Analytical Biochemistry
Yosvany López, Abdollah Dehzangi, Sunil Pranit Lal, Ghazaleh Taherzadeh, Jacob Michaelson, Abdul Sattar, Tatsuhiko Tsunoda, Alok Sharma
Post-Translational Modification (PTM) is a biological reaction which contributes to diversify the proteome. Despite many modifications with important roles in cellular activity, lysine succinylation has recently emerged as an important PTM mark. It alters the chemical structure of lysines, leading to remarkable changes in the structure and function of proteins. In contrast to the huge amount of proteins being sequenced in the post-genome era, the experimental detection of succinylated residues remains expensive, inefficient and time-consuming...
March 28, 2017: Analytical Biochemistry
Sharik R Khan, Andrei Kuzminov
We showed before that long linear DNA molecules containing single-strand interruptions and undergoing pulsed-field gel electrophoresis (PFGE) tend to break into subfragments (electrophoretic nick instability). Here we show that circular chromosomal DNA with single-strand interruptions remains in the wells during PFGE. This means that the presence of nicks in immobile circular DNA is not enough to break this DNA during PFGE. In other words, under the conditions of our study, the artifactual conversion of nicks into double-strand breaks that we detect in linear DNA does not contribute to the overall level of chromosomal fragmentation, as measured by PFGE...
March 27, 2017: Analytical Biochemistry
Lu Chen, Hui-Ting Lian, Xiang-Ying Sun, Bin Liu
A novel electrochemical sensor was presented for the determination of L-5-hydroxytryptophan (L-5-HTP) based on a graphene-chitosan molecularly imprinted film modified on the surface of glassy carbon electrode (GR-MIP/GCE). The morphology and composition of the imprinted film were observed in field emission scanning electron microscopy (FESEM), raman spectroscopy and fourier transform infrared (FTIR). The properties of the sensor were evaluated by electrochemical techniques. Under the optimal conditions, the peak currents of L-5-HIP were found to be linear in the concentration range of 0...
March 19, 2017: Analytical Biochemistry
Xingping Wu, Ailin Zhu, Xiaowu Pang, Guiqin Xie, Xinbin Gu
We describe a simple method to accurately detect and quantify both Pten mutation and allele-specific loss using allele-specific PCR analysis. Our approach used a heterozygous genomic DNA with one wild-type and one mutant Pten allele as a reference at a single concentration to calculate the percent ratio of the wild-type Pten gene for the detection of allele-specific gene loss. With a standard curve, ratios from PCR data were used to quantitate the wild-type Pten allele copy number loss in tumor specimens. We demonstrate the utility of our approach to calculate allele-specific Pten loss during tumor progression and show that our approach generates quantitative data that are comparable to those obtained from digital droplet PCR...
March 18, 2017: Analytical Biochemistry
Dal-Hoe Koo, Bhupendra Singh, Jiming Jiang, Bernd Friebe, Bikarm S Gill, Paul D Chastain, Upender Manne, Hemant K Tiwari, Keshav K Singh
Somatic human cells contain thousands of copies of mitochondrial DNA (mtDNA). In eukaryotes, natural transfer of mtDNA into the nucleus generates nuclear mitochondrial DNA (NUMT) copies in eukaryotes. We name this phenomenon as "numtogenesis". Numtogenesis is a well-established evolutionary process reported in various sequenced eukaryotic genomes. We have established a molecular tool to rapidly detect and analyze NUMT insertions in whole genomes. To date, NUMT analyses depend on deep genome sequencing combined with comprehensive computational analyses of the whole genome...
March 18, 2017: Analytical Biochemistry
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