6,636 results match your criteria BioTechniques[Journal]
Biotechniques 2018 May;64(5):193
Head of Open Access Publishing; Future Science Group, London, UK.
Biotechniques 2018 May;64(5):203-210
Department of Biomedical Engineering, College of Engineering, Ohio State University, 460 W 12th Ave, 504 Biomedical Research Tower, Columbus, OH 43210, USA.
Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Read More
Biotechniques 2018 May;64(5):225-230
Institute of Clinical Molecular Biology, Christian-Albrechts-Universität zu Kiel, Schleswig-Holstein, D-24105 Kiel, Germany.
DNA can enter the blood circulation from living cells by extracellular vesicles or at cell death, and pass into urine through the kidney barrier. Urine can be collected non-invasively, making it an interesting source of cell-free DNA (cfDNA) for research studies and ultimately for clinical diagnostics. However, there is currently a lack of data on the quantity and variability of cfDNA in urine. Read More
Biotechniques 2018 May;64(5):211-217
Key Laboratory for Medical Virology, National Health & Family Planning Commission, National Institute for Viral Disease Control & Prevention, Chinese Center for Disease Control & Prevention, Beijing 102206, China.
Single nucleotide polymorphisms (SNPs) play an important role in susceptibility to complex diseases, treatment efficacy and adverse drug responses. Conventional methods to detect SNPs are usually based on PCR or DNA sequencing, which are typically time-consuming and require sophisticated equipment. In this proof-of-concept study, a probe-directed recombinase amplification (PDRA) assay was developed to detect the A1298C polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR). Read More
Biotechniques 2018 May;64(5):197-201
Future Science Group, Unitec House, 2 Albert Place, London, N3 1QB.
Protein analysis is crucial to elucidating the function of proteins and understanding the impact of their presence, absence and alteration. This is key to advancing knowledge about diseases, providing the opportunity for biomarker discovery and development of therapeutics. In this issue of Tech News, Nawsheen Boodhun explores the various means of protein analysis. Read More
Biotechniques 2018 May;64(5):194-196
Northwestern University Feinberg School of Medicine, IL, USA.
Dr Joshua Z Rappoport, PhD, speaks to Nawsheen Boodhun, Managing Editor. Rappoport completed his bachelor's degree in Biology at Brown University (RI, USA). He then went on to earn a PhD from the Program in Mechanisms of Disease and Therapeutics at the Mount Sinai School of Medicine Graduate School of Biological Sciences of New York University (USA). Read More
Biotechniques 2018 May 25;64(5):231-234. Epub 2018 Apr 25.
Department of Biomedical Science & Engineering, Gwangju Institute of Science & Technology, Republic of Korea.
Most conventional hypoxic cell culture systems undergo reoxygenation during experimental manipulations, resulting in undesirable effects including the reduction of cell viability. A lid device was developed herein for conventional cell culture dishes to resolve this limitation. The integration of multilayered microfluidic channels inside a thin membrane was designed to prevent the reoxygenation caused by reagent infusion and automatically control the oxygen level. Read More
Biotechniques 2018 May 19;64(5):219-224. Epub 2018 Apr 19.
Key Laboratory of Plant Genetics & Molecular Breeding, Zhoukou Normal University, Zhoukou, 466001, PR China.
Yeast two-hybrid systems are powerful tools for analyzing interactions between proteins. Vector construction is an essential step in yeast two-hybrid experiments, which require bait and prey plasmids. In this study, we modified the multiple cloning site sequence of the yeast plasmid pGADT7 by site-directed mutagenesis PCR to generate the pGADT7-In vector, which resulted in an easy and rapid method for constructing yeast two-hybrid vectors using the In-Fusion cloning technique. Read More
Biotechniques 2018 04;64(4):185-186
Biotechniques 2018 04;64(4):141
Biotechniques 2018 04;64(4):187-188
Biotechniques 2018 04;64(4):184
Biotechniques 2018 Apr;64(4):153-161
Center for Bioscience Research and Education, Utsunomiya University, Tochigi, Japan.
The bimolecular fluorescence complementation (BiFC) assay was developed as a tool for the visualization of protein-protein interactions in living cells. To date, many types of BiFC systems with distinct colors have been developed. Most of the colors in the visible spectrum have been used in BiFC assays, with the exception of orange. Read More
Biotechniques 2018 Apr;64(4):128-130
Biotechniques 2018 Apr;64(4):143-146
Future Science Group, Unitec House, 2 Albert Place, London, N3 1QB.
The ability to elucidate the structure and function of biological molecules holds great importance in a variety of domains. This includes prospects to tackle public health concerns, identification of new drug targets and therapeutic agents. In this issue of Tech News, Nawsheen Boodhun explores techniques used to understand macromolecules. Read More
Biotechniques 2018 Apr;64(4):163-169
Biology Department, University Massachusetts Amherst, Amherst, MA 01003, USA.
The diversity of spider behavior and sensory systems provides an excellent opportunity for comparative studies of the relationship between the brain and behavior. However, the morphology of spiders poses a challenge for histologists since the spider cephalothorax contains heterogeneous tissues and has both tough external and internal sclerotized regions. Unlike the heads of insects, the cephalothorax is highly pressurized, which can cause tissues to shift during processing and can reduce tissue cohesion in thick sections. Read More
Biotechniques 2018 Apr;64(4):177-179
Arcadia University, Department of Chemistry & Physics, Glenside, PA, USA.
Real-time PCR (qPCR) is widely used in the life sciences. For quantifying DNA, a standard curve is required. Common methods for standard development are time consuming, costly, necessitate a specific skill set, and pose a contamination risk. Read More
Biotechniques 2018 Apr;64(4):170-176
Counterterrorism & Forensic Science Research Unit, Federal Bureau of Investigation Laboratory Division, 2501 Investigation Parkway, Quantico, VA 22135, USA.
A simple method for extracting protein from human anagen (i.e., actively growing hair stage) head hairs was developed in this study for cases of limited sample availability and/or studies of specific micro-features within a hair. Read More
Biotechniques 2018 Apr;64(4):181-183
Targeted Therapeutic Drug Discovery & Development Program, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA.
The use of AlphaScreen detection has allowed researchers to examine a wide variety of molecular interactions for use in high-throughput screening. However, the cost of Alpha reagents can often be prohibitory for extended screening campaigns or for young investigators with limited funding. To reduce assay costs, many labs have focused on miniaturization, while there have been limited efforts to scale down Alpha reagents. Read More
Biotechniques 2018 Apr;64(4):139
Head of Open Access Publishing, Future Science Group, Unitec House, 2 Albert Place, London, UK.
Biotechniques 2018 Feb 1;64(2):69-80. Epub 2018 Feb 1.
State Key Laboratory of Bioelectronics, Southeast University, 210096, Nanjing, China.
This paper presents a new rolling circle amplification (RCA) technique using stem-loop primers (SLP). The technique enables detection of target DNA by either linear or exponential amplification (SLP-lRCA and SLP-eRCA) in both liquid and solid phases. For solid-phase detection, SLP-eRCA detects nucleic acids in four steps: (1) covalently immobilize an array of capture probes (CP) on a solid support; (2) hybridize the CP array with the DNA sample; (3) incubate the CP array with an RCA reaction containing two SLPs; (4) image the CP array. Read More
Biotechniques 2018 Feb 1;64(2):59-68. Epub 2018 Feb 1.
The Biosystems and Biomaterials Division, The National Institute of Standards and Technology, 100 Bureau Drive, MD 20899, Gaithersburg.
We compared different methods (absorbance, fluorescent dye-binding, and digital PCR) for measuring the concentrations of human genomic DNA from cultured cells and absorbance measurements of a synthetic DNA oligonucleotide. NIST Standard Reference Material (SRM) 2082, a pathlength absorbance standard, was used to benchmark the absorbance measurements done with microvolume spectrophotometers and a microvolume plate reader. Control absorbance values were measured on a high accuracy spectrophotometer and a NIST calibrated pathlength cuvette. Read More
Biotechniques 2018 Feb 1;64(2):52-58. Epub 2018 Feb 1.
Discipline of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Private Bag X01, 3209, Scottsville, South Africa.
The communication between nonmyogenic cells, such as macrophages and fibroblasts, and myoblasts is crucial for successful skeletal muscle repair. In vitro co-culture methods can be used to increase our understanding of these cellular interactions; however, current protocols are restricted to two, often physically separate, cell populations. Here, we demonstrate a novel, inexpensive in vitro triple co-culture method that facilitates the co-culture of at least three cell populations with some degree of cell-cell contact. Read More
Biotechniques 2018 Feb 1;64(2):45-51. Epub 2018 Feb 1.
Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, 43124, Parma, Italy.
FTA® technologies provide the most reliable method for DNA extraction. Although FTA technologies have been widely used for genetic analysis, there is no literature on their use for epigenetic analysis yet. We present for the first time, a simple method for quantitative methylation assessment based on sperm cells stored on Whatman FTA classic cards. Read More
Biotechniques 2018 Mar 1;64(3):118-124. Epub 2018 Mar 1.
Division of Animal Science, National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA.
Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. Read More
Biotechniques 2018 Mar 1;64(3):110-116. Epub 2018 Mar 1.
Russian Academy of Sciences, Miklukho-Maklaya 16/10, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997, Moscow, Russia.
Despite considerable success studying glycan-binding proteins using printed glycan arrays (PGAs), unambiguous quantitation of spot intensities by fluorescent readers remains a challenge. The main obstacles are the varying spot shape and size and in-spot fluorescence distribution caused by uneven drying of the printed drops. Two methods have been suggested for solving this problem: using polymeric glycoconjugates, which makes it possible to equalize the physicochemical properties (hydrophobicity, charge, and size) of different glycans, and applying a glycan solution on a slide coated with a thin oil mask, which hinders evaporation of the drop. Read More
Biotechniques 2018 Mar 1;64(3):101-109. Epub 2018 Mar 1.
Department of Physiology, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Metabolite diffusion in tissues produces gradients and heterogeneous microenvironments that are not captured in standard 2D cell culture models. Here we describe restricted exchange environment chambers (REECs) in which diffusive gradients are formed and manipulated on length scales approximating those found in vivo. In REECs, cells are grown in 2D in an asymmetric chamber (<50 μL) formed between a coverglass and a glass bottom cell culture dish separated by a thin (~100 μm) gasket. Read More
Biotechniques 2018 Mar 1;64(3):96-99. Epub 2018 Mar 1.
3D printed biomaterials are increasingly used in cell cultures and drug screens. Given the ease of creating artificial tissues, will this technique revolutionize biomedicine and organ implants in the future? Read More
Biotechniques 2018 Jan 1;64(1):27-29. Epub 2018 Jan 1.
School of Life and Environmental Sciences, Charles Perkins Centre, The University of Sydney.
Research into cellular metabolism has become more high-throughput, with typical cell-culture experiments being performed in multiwell plates (microplates). This format presents a challenge when trying to collect gaseous products, such as carbon dioxide (CO2), which requires a sealed environment and a vessel separate from the biological sample. To address this limitation, we developed a gas trapping protocol using perforated plastic lids in sealed cell-culture multiwell plates. Read More
Biotechniques 2018 Jan 1;64(1):24-26. Epub 2018 Jan 1.
Institute of Biophysics and Physical Biochemistry, University of Regensburg, Regensburg, Germany.
Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. Read More
Biotechniques 2018 Jan 1;64(1):21-23. Epub 2018 Jan 1.
Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Previously, we reported a method for facile purification of oligonucleotides labeled with hydrophobic dyes, based on the solubility difference between the hydrophilic DNA and unreacted dye. Here, we present a new purification method applicable to any dye regardless of its hydrophobicity. We exploited the population shift of a fluorescent dye in a low-pH aqueous solution from its anionic form toward its neutral form. Read More
Biotechniques 2018 Jan 1;64(1):13-19. Epub 2018 Jan 1.
Department of Biomedical Engineering, University of Wisconsin, Madison, WI.
Cellular heterogeneity within the tissue microenvironment may underlie chemotherapeutic resistance and response, enabling tumor evolution; however, this heterogeneity it is difficult to characterize. Here, we present a new approach-pathology-guided micropunching (PGM)-that enables identification and characterization of heterogeneous foci identified in viable human and animal model tissue slices. This technique consists of live-cell tissue labeling using fluorescent antibodies/small molecules to identify heterogeneous foci (e. Read More
Biotechniques 2017 Dec 1;63(6):281-283. Epub 2017 Dec 1.
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Here, we describe a method for obtaining thin cross sections of Arabidopsis thaliana roots without fixation and embedding. Roots were grown in pinholes made in a solidified growth medium, and cross sections were prepared without pretreatment. Using this method, we detected unique distributions of two polar-localized proteins-green fluorescent protein (GFP)-tagged BOR1 and NIP5;1-with less sample preparation time than conventional methods. Read More
Biotechniques 2017 Dec 1;63(6):275-280. Epub 2017 Dec 1.
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI.
Accurately characterizing cellular subpopulations is essential for elucidating the mechanisms underlying normal and pathological biology. Isolation of specific cell types can be accomplished by labeling unique cell-associated proteins with fluorescent antibodies. Cell fixation is commonly used to prepare these samples and allow for long-term storage, but this poses challenges for subsequent protein analysis. Read More
Biotechniques 2017 Dec 1;63(6):267-274. Epub 2017 Dec 1.
Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC.
Cell migration, which is central to a wide variety of life processes, involves integration of the extracellular matrix (ECM) with the internal cytoskeleton and motor proteins via receptors spanning the plasma membrane. Cell migration can be induced by a variety of signals, including gradients of external soluble molecules, differences in ECM composition, or electrical gradients. Current in vitro methods to study cell migration only test one substrate at a time. Read More
Biotechniques 2017 Dec 1;63(6):261-266. Epub 2017 Dec 1.
Avicor Ltd., Szeged, Hungary.
Synthetic DNA has been used as an authentication code for a diverse number of applications. However, existing decoding approaches are based on either DNA sequencing or the determination of DNA length variations. Here, we present a simple alternative protocol for labeling different objects using a small number of short DNA sequences that differ in their melting points. Read More
Biotechniques 2017 Dec 1;63(6):253-260. Epub 2017 Dec 1.
School of Materials, The University of Manchester, Manchester, UK.
Continuous optimization of in vitro analytical techniques is ever more important, especially given the development of new materials for tissue engineering studies. In particular, isolation of cellular components for downstream applications is often hindered by the presence of biomaterials, presenting a major obstacle in understanding how cell-matrix interactions influence cell behavior. Here, we describe an approach for western blot analysis of cells that have been encapsulated in self-assembling peptide hydrogels (SAPHs), which highlights the need for complete solubilization of the hydrogel construct. Read More
Biotechniques 2017 Nov 1;63(5):236-237. Epub 2017 Nov 1.
Pharmaceutical Design and Discovery Group (PharmDD), Faculty of Pharmacy, University of Helsinki.
Biotechniques 2017 Nov 1;63(5):235. Epub 2017 Nov 1.
Biotechniques 2017 Nov 1;63(5):230-233. Epub 2017 Nov 1.
Division of Dermatology, University of Ottawa, Ottawa, Ontario, Canada.
Immunostaining of non-adherent cells is commonly performed after adhesion of cells onto microscope slides either using cytocentrifugation or with the help of charged coating substrates. These techniques, however, require either specialized equipment or significant preparation time. Here, we describe a method for immunofluorescent staining of lymphocytes within multi-well culture plates, where cells suspended in phosphate buffered saline (PBS) are adhered to either the plastic well bottom or glass coverslips by gravity sedimentation. Read More
Biotechniques 2017 Nov 1;63(5):227-229. Epub 2017 Nov 1.
Advanced Research Center on Electronic Systems (ARCES) for Information and Communication Technologies "E. De Castro," University of Bologna, Italy; Department of Computer Science and Engineering (DISI), University of Bologna, Italy.
Cancer 3-D spheroids are widely used to test drugs and radiotherapy treatments. These 3-D cell clusters range from tens to hundreds of micrometers in size, with shapes that typically differ from a perfect sphere. Change in spheroid volume is one of the most important parameters for evaluating treatment efficacy, and using light sheet fluorescence microscopes (LSFM), optical sections of samples in that size range can be obtained. Read More
Biotechniques 2017 Nov 1;63(5):221-226. Epub 2017 Nov 1.
Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY.
Quantitative analysis of next-generation sequencing (NGS) data requires discriminating duplicate reads generated by PCR from identical molecules that are of unique origin. Typically, PCR duplicates are identified as sequence reads that align to the same genomic coordinates using reference-based alignment. However, identical molecules can be independently generated during library preparation. Read More
Biotechniques 2017 Nov 1;63(5):215-220. Epub 2017 Nov 1.
Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, KS.
Analysis of mouse behavior often requires expensive equipment and transfer of the mice to new test environments, which could trigger confounding behavior alterations. Here, we describe a system for tracking mouse behavior in home cages using a low-cost USB webcam and free software (Fiji and wrMTrck). We demonstrate the effectiveness of this method by tracking differences in distance traveled, speed, and movement tracks between wild-type mice and amyotrophic lateral sclerosis (ALS) model mice (SOD1G93A). Read More
Biotechniques 2017 Nov 1;63(5):205-214. Epub 2017 Nov 1.
Institute of Virology, University of Ulm, Ulm, Germany.
For immunological research on the human cytomegalovirus (HCMV), a virus that combines the broad cell tropism of clinical isolates, efficient replication in cell culture, the complete set of MHC-I modulator genes, and suitability for genetic engineering is desired. Here, we aimed to generate a genetically complete derivative of HCMV strain TB40/E as a bacterial artificial chromosome (BAC) with a self-excisable BAC cassette. The BAC cassette was inserted into the US2-US6 gene region (yielding TB40-BACKL7), relocated into the UL73/UL74 region with modifications that favor excision of the BAC cassette during replication in fibroblasts, and finally the US2-US6 region was restored, resulting in BAC clone TB40-BACKL7-SE When this BAC clone was transfected into fibroblasts at efficiencies >0. Read More