scholarly journals One-step split GFP staining for sensitive protein detection and localization in mammalian cells

BioTechniques ◽  
2010 ◽  
Vol 49 (4) ◽  
pp. 727-736 ◽  
Author(s):  
Lara Kaddoum ◽  
Eddy Magdeleine ◽  
Geoffrey S. Waldo ◽  
Etienne Joly ◽  
Stéphanie Cabantous
Keyword(s):  
Mammalian Cells ◽  
Protein Detection ◽  
One Step ◽  
Detection And Localization

scholarly journals One step DNA amplification of mammalian cells in picoliter microwell arrays

RSC Advances ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 2865-2869 ◽  
Author(s):  
Wenwen Liu ◽  
Zhao Li ◽  
Yuanjie Liu ◽  
Qingquan Wei ◽  
Yong Liu ◽  
...  
Keyword(s):  
Single Cell ◽  
Mammalian Cells ◽  
Dna Amplification ◽  
Single Copy ◽  
Microwell Array ◽  
One Step

One-step PCR of a single cell in a picoliter microwell array was developed and applied to detect a target with the sensitivity of a single copy.

scholarly journals One-step high-throughput assay for quantitative detection of β-galactosidase activity in intact Gram-negative bacteria, yeast, and mammalian cells

BioTechniques ◽  
2006 ◽  
Vol 40 (4) ◽  
pp. 433-440 ◽  
Author(s):  
Faustino Vidal-Aroca ◽  
Michele Giannattasio ◽  
Elisa Brunelli ◽  
Alessandro Vezzoli ◽  
Paolo Plevani ◽  
...  
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Quantitative Detection ◽  
Gram Negative Bacteria ◽  
Galactosidase Activity ◽  
Gram Negative ◽  
One Step ◽  
High Throughput Assay

scholarly journals Fast purification of recombinant monomeric amyloid-β from E. coli and amyloid-β-mCherry aggregates from mammalian cells

2020 ◽  
Author(s):  
Amberley D Stephens ◽  
Meng Lu ◽  
Gabriele S Kaminski Schierle
Keyword(s):  
Ion Exchange ◽  
Mammalian Cells ◽  
Inclusion Bodies ◽  
Amyloid Β ◽  
Time Frame ◽  
Purification Method ◽  
Vast Array ◽  
E Coli ◽  
Freeze Dried ◽  
One Step

The Alzheimer's disease related peptide, Amyloid-beta (Aβ)1-40 and 1-42, has proven difficult to be purified as a recombinant monomeric protein due its expression in E. coli leading to the formation of insoluble inclusion bodies and its tendency to quickly form insoluble aggregates. A vast array of methods have been used so far, yet many have pitfalls, such as the use of tags for ease of Aβ isolation, the formation of Aβ multimers within the time frame of extraction or the need to reconstitute Aβ from a freeze dried state. Here, we present a rapid protocol to produce highly pure and monomeric recombinant Aβ using a one-step ion exchange purification method and to label the peptide using a maleimide dye. The solublisation and purification steps take only three hours. We also present a protocol for the isolation of Aβ-mCherry from mammalian cells.

scholarly journals Construction and validation of EGFP-expressingStaphylococcus aureusclinical strains for adhesion and internalization assays on epithelial cells

2019 ◽  
Author(s):  
Sana Charaoui-Boukerzaza ◽  
M. Fedy Morgene ◽  
Josselin Rigaill ◽  
Estelle Audoux ◽  
Zhiguo He ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Epithelial Cells ◽  
Mammalian Cells ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Egfp Expression ◽  
Confocal Laser ◽  
Wild Type ◽  
Clinical Strains ◽  
One Step

ABSTRACTBackgroundStaphylococcus aureusis both a major pathogen and a commensal bacterium in humans. It is able to adhere at the surface of epithelial cells of the anterior nares and can trigger its internalization inside these non-professional phagocytic cells. To better understand the interactions of clinical isolates with keratinocytes in the anterior nares, we developed and validated a one-step protocol expressing enhanced green fluorescent protein (EGFP) inS. aureusclinical strains with the aim to study adhesion to and internalization into mammalian cells.MethodsTwentyS. aureusclinical isolates belonging to clonal complexes 5, 8, 30, 45, 398 were selected for one-step transformation protocol with the EGFP-encoding plasmid pBSU101. EGFP expression was analysed by flow cytometry and confocal microscopy. Wild type and isogenic EGFP-expressing strains were compared for adhesion and internalization levels by using the HaCaT cell model.ResultsTransformation was achieved in all theS. aureusstrains regardless of their genetic background. The flow cytometry analysis showed that the mean proportion of EGFP-expressing bacteria was 97.2% (± 2.1) after 4h of incubation. Adhesion and internalization levels were similar in wild-type and isogenic EGFP-expressingS. aureusstrains. Confocal laser scanning microscopy confirmed that EGFP-expressingS. aureusbacteria could be easily identified inside HaCaT keratinocytes.ConclusionThis study reports an efficient protocol for expressing EGFP inS. aureusclinical strains and demonstrates that these EGFP-expressing strains are suitable for adhesion and internalization assays using HaCaT cells, which allows to perform static and dynamicin vitrostudies ofS. aureuscolonization.

scholarly journals One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: A protocol to study infection & gene expression during ZIKV infection

2019 ◽  
Author(s):  
Ricardo Vieira Araujo ◽  
Fabiana Feitosa-Suntheimer ◽  
Alexander S. Gold ◽  
Berlin Londono-Renteria ◽  
Tonya Michelle Colpitts
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Infected Mosquito ◽  
Zikv Infection ◽  
Rna Detection ◽  
High Background ◽  
Qrt Pcr ◽  
One Step ◽  
Nonspecific Amplification ◽  
Lower Background

Abstract Background: Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is qRT-PCR, yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito RNA. In this work we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing for confident detection.Methods: Our new oligo design to detect ZIKV RNA included in silicoanalysis of both viral and mosquito (Ae. aegyptiand Ae. albopictus)genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedesgenomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes.Standard curves constructed with in vitrotranscribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay.Results: Finally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegyptiimmune response to virus infection.Conclusions: The experimental approach to detect ZIKV RNA in Aedes aegyptipresented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.

scholarly journals A one-step tRNA-CRISPR system for genome-wide genetic interaction mapping in mammalian cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yulei Zhao ◽  
Kathrin Tyrishkin ◽  
Calvin Sjaarda ◽  
Prem Khanal ◽  
Jeff Stafford ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Drug Targets ◽  
Genetic Interaction ◽  
Genetic Interactions ◽  
Cancer Treatments ◽  
Genome Wide ◽  
A Genome ◽  
High Efficient ◽  
One Step ◽  
Screening Accuracy

Abstract Mapping genetic interactions in mammalian cells is limited due to technical obstacles. Here we describe a method called TCGI (tRNA-CRISPR for genetic interactions) to generate a high-efficient, barcode-free and scalable pairwise CRISPR libraries in mammalian cells for identifying genetic interactions. We have generated a genome- wide library to identify genes genetically interacting with TAZ in cell viability regulation. Validation of candidate synergistic genes reveals the screening accuracy of 85% and TAZ-MCL1 is characterized as combinational drug targets for non-small cell lung cancer treatments. TCGI has dramatically improved the current methods for mapping genetic interactions and screening drug targets for combinational therapies.

scholarly journals Sensitive Detection of C-Reactive Protein by One-Step Method Based on a Waveguide-Mode Sensor

Sensors ◽  
2020 ◽  
Vol 20 (11) ◽  
pp. 3195 ◽  
Author(s):  
Hiroki Ashiba ◽  
Chiaki Oyamada ◽  
Kazuya Hosokawa ◽  
Koji Ueno ◽  
Makoto Fujimaki
Keyword(s):  
Waveguide Mode ◽  
Protein Detection ◽  
Sensor Signal ◽  
C Reactive Protein ◽  
Step Method ◽  
Reactive Protein ◽  
Sensor Signals ◽  
Evanescent Light ◽  
One Step ◽  
Surface Performance

One-step biosensing methods enable the quick and simplified detection of biological substances. In this study, we developed a sensitive one-step method on the basis of a waveguide-mode sensor, which is an optical sensor utilizing waveguide-mode resonance and evanescent light. Streptavidin-conjugated and gold-nanoparticle-conjugated antibodies were reacted with a target substance and applied onto a biotinylated sensing plate. The target substance was detected by observing changes in sensor signals caused by binding the immunocomplex to the sensing surface. Performance of the developed one-step method was examined using a C-reactive protein (CRP) as a target substance. A sensor signal corresponding to the concentration of CRP was obtained. The minimal detectable CRP concentration of the developed method was 10 pM. The developed method greatly simplifies quantitative protein detection without reducing sensitivity.

scholarly journals Ratiometric electrochemical proximity assay for sensitive one-step protein detection

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Kewei Ren ◽  
Jie Wu ◽  
Feng Yan ◽  
Huangxian Ju
Keyword(s):  
Protein Detection ◽  
One Step

scholarly journals Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells

2004 ◽  
Vol 383 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Irina V. KOREEN ◽  
Wafaa A. ELSAYED ◽  
Yu J. LIU ◽  
Andrew L. HARRIS
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Expression System ◽  
Physiological Processes ◽  
Messenger Molecules ◽  
One Step ◽  
Cellular Control

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10–20 μg of pure connexin protein from 2.5×108 HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)–agarose make this affinity tagging and purification procedure easily applicable to other proteins.

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