Assay systems

  • Article
    | Open Access

    Alternative splicing is regulated by multiple mechanisms. Here the authors employed designed splice site libraries and massively parallel reporter assays to dissect the regulatory complexity and cell-to-cell variability of splicing decisions and to build accurate predictive models.

    • Martin Mikl
    • , Amit Hamburg
    • , Yitzhak Pilpel
    •  & Eran Segal
  • Article
    | Open Access

    Epitope tags are widely used in various applications, but often lack versatility. Here, the authors introduce a small, alpha helical tag, which is recognized by a high affinity nanobody and can be used in a range of different applications, from protein purification to super-resolution imaging and in vivo detection of proteins.

    • Hansjörg Götzke
    • , Markus Kilisch
    • , Markel Martínez-Carranza
    • , Shama Sograte-Idrissi
    • , Abirami Rajavel
    • , Thomas Schlichthaerle
    • , Niklas Engels
    • , Ralf Jungmann
    • , Pål Stenmark
    • , Felipe Opazo
    •  & Steffen Frey
  • Article
    | Open Access

    Bacterial allosteric transcription factors can sense and respond to a variety of small molecules. Here the authors present CaT-SMelor which uses Cas12a and allosteric transcription factors to detect small molecules in the nanomolar range.

    • Mindong Liang
    • , Zilong Li
    • , Weishan Wang
    • , Jiakun Liu
    • , Leshi Liu
    • , Guoliang Zhu
    • , Loganathan Karthik
    • , Man Wang
    • , Ke-Feng Wang
    • , Zhong Wang
    • , Jing Yu
    • , Yuting Shuai
    • , Jiaming Yu
    • , Lu Zhang
    • , Zhiheng Yang
    • , Chuan Li
    • , Qian Zhang
    • , Tong Shi
    • , Liming Zhou
    • , Feng Xie
    • , Huanqin Dai
    • , Xueting Liu
    • , Jingyu Zhang
    • , Guang Liu
    • , Ying Zhuo
    • , Buchang Zhang
    • , Chenli Liu
    • , Shanshan Li
    • , Xuekui Xia
    • , Yaojun Tong
    • , Yanwen Liu
    • , Gil Alterovitz
    • , Gao-Yi Tan
    •  & Li-Xin Zhang
  • Article
    | Open Access

    Monitoring protein-protein interactions via bimolecular fluorescence complementation is often limited by the slow kinetics and irreversibility of the complementation. Here the authors introduce a fluorescent reporter for real-time monitoring of reversible interactions based on complementation and binding of an exogenous chromophore.

    • Alison G. Tebo
    •  & Arnaud Gautier
  • Article
    | Open Access

    The range of chemicals detectable by cell-free systems is still limited. Here the authors combine metabolic cascades with transcription factor networks to detect small molecules in complex environments.

    • Peter L. Voyvodic
    • , Amir Pandi
    • , Mathilde Koch
    • , Ismael Conejero
    • , Emmanuel Valjent
    • , Philippe Courtet
    • , Eric Renard
    • , Jean-Loup Faulon
    •  & Jerome Bonnet
  • Article
    | Open Access

    Simultaneous quantification of DNA, RNA and protein at the single cell level has not yet been possible. Here the authors introduce a molecular labelling and detection strategy to quantify synthesis of these biomolecules and couple it to transient cell states through parallel quantification of state-dependent biomolecules.

    • Samuel C. Kimmey
    • , Luciene Borges
    • , Reema Baskar
    •  & Sean C. Bendall
  • Article
    | Open Access

    Isothermal DNA amplification techniques are useful for diagnostic applications in place of traditional PCR. Here the authors describe CRISDA, which combines CRISPR–Cas9 with strand displacement amplification and exhibits attomolar sensitivity and single-nucleotide specificity in DNA detection.

    • Wenhua Zhou
    • , Li Hu
    • , Liming Ying
    • , Zhen Zhao
    • , Paul K. Chu
    •  & Xue-Feng Yu
  • Article
    | Open Access

    Single-cell detection methods are limited by the trade-off between flow rate and measurement precision. Here the authors introduce active loading, an optically triggered microfluidic system to concentrate diluted cell samples, which reduces clogging and decreases processing time in single-cell assays.

    • Nicholas L. Calistri
    • , Robert J. Kimmerling
    • , Seth W. Malinowski
    • , Mehdi Touat
    • , Mark M. Stevens
    • , Selim Olcum
    • , Keith L. Ligon
    •  & Scott R. Manalis
  • Article
    | Open Access

    Membrane bilayers are made up of a myriad of different lipids that affect membrane proteins, but identifying those specific lipid requirements remains a challenge. Here authors present an engineered thermal-shift screen which reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins.

    • Emmanuel Nji
    • , Yurie Chatzikyriakidou
    • , Michael Landreh
    •  & David Drew
  • Article
    | Open Access

    Five validation pillars have been proposed to verify the specificity of research antibodies. Here the authors screen 6,000 antibodies from the Human Protein Atlas with these methods to provide an antibody validation resource for providers and users.

    • Fredrik Edfors
    • , Andreas Hober
    • , Klas Linderbäck
    • , Gianluca Maddalo
    • , Alireza Azimi
    • , Åsa Sivertsson
    • , Hanna Tegel
    • , Sophia Hober
    • , Cristina Al-Khalili Szigyarto
    • , Linn Fagerberg
    • , Kalle von Feilitzen
    • , Per Oksvold
    • , Cecilia Lindskog
    • , Björn Forsström
    •  & Mathias Uhlen
  • Article
    | Open Access

    Current methods to unveil protein–protein and protein–RNA interactions often involve long, costly procedures. Here the authors present a rapid yeast 2- and 3-hybrid screening pipeline to detect interactions within protein libraries or between protein libraries and RNA fragment pools.

    • Jae-Seong Yang
    • , Mireia Garriga-Canut
    • , Nele Link
    • , Carlo Carolis
    • , Katrina Broadbent
    • , Violeta Beltran-Sastre
    • , Luis Serrano
    •  & Sebastian P. Maurer
  • Article
    | Open Access

    Isolation of circulating tumor cells (CTCs) allows for non-invasive disease monitoring and characterization. Here the authors describe an alternative CTC isolation method based on the ability of the malaria rVAR2 protein to specifically bind oncofetal chondroitin sulfate, which is expressed by all cancer cells

    • Mette Ø. Agerbæk
    • , Sara R. Bang-Christensen
    • , Ming-Hsin Yang
    • , Thomas M. Clausen
    • , Marina A. Pereira
    • , Shreya Sharma
    • , Sisse B. Ditlev
    • , Morten A. Nielsen
    • , Swati Choudhary
    • , Tobias Gustavsson
    • , Poul H. Sorensen
    • , Tim Meyer
    • , David Propper
    • , Jonathan Shamash
    • , Thor G. Theander
    • , Alexandra Aicher
    • , Mads Daugaard
    • , Christopher Heeschen
    •  & Ali Salanti
  • Article
    | Open Access

    Current sensors for intracellular H2O2 are not sensitive enough to detect all biologically relevant H2O2 fluctuations. Here the authors develop a peroxiredoxin-2-based FRET probe that is sensitive enough to measure changes in H2O2 concentration in response to the cancer therapeutic piperlongumine.

    • Troy F. Langford
    • , Beijing K. Huang
    • , Joseph B. Lim
    • , Sun Jin Moon
    •  & Hadley D. Sikes
  • Article
    | Open Access

    Cancer patients exhibit specific sensitivities toward drug combinations that cannot be easily predicted. Here the authors setup a microfluidic platform that allows testing of multiple drug combinations correctly predicting sensitivity in vivo and they use it on patients biopsies to define effective drugs.

    • Federica Eduati
    • , Ramesh Utharala
    • , Dharanija Madhavan
    • , Ulf Peter Neumann
    • , Thomas Longerich
    • , Thorsten Cramer
    • , Julio Saez-Rodriguez
    •  & Christoph A. Merten
  • Article
    | Open Access

    The Trioplex real-time RT-PCR assay was developed for detection of Zika virus infections in areas with dengue and chikungunya transmission. Here, Santiago et al. describe the optimization and clinical performance of the assay, showing high sensitivity for detection and differentiation of the three viruses.

    • Gilberto A. Santiago
    • , Jesús Vázquez
    • , Sean Courtney
    • , Katia Y. Matías
    • , Lauren E. Andersen
    • , Candimar Colón
    • , Angela E. Butler
    • , Rebecca Roulo
    • , John Bowzard
    • , Julie M. Villanueva
    •  & Jorge L. Muñoz-Jordan
  • Article
    | Open Access

    Detecting metastatic cells in tumor/lymph node samples of breast cancer patients is extremely important for diagnosis. Here the authors develop a microelectronic biochip that detect the presence of invasive/metastatic cells in unprocessed biopsies and performs better than the current gold standards.

    • Mohammad Saeid Nikshoar
    • , Mohammad Ali Khayamian
    • , Saeid Ansaryan
    • , Hassan Sanati
    • , Milad Gharooni
    • , Leila Farahmand
    • , Farshad Rezakhanloo
    • , Keivan Majidzadeh-A
    • , Parisa Hoseinpour
    • , Shahrzad Dadgari
    • , Leila Kiani-M
    • , Mohammad Saqafi
    • , Masoumeh Gity
    •  & Mohammad Abdolahad
  • Article
    | Open Access

    The interrogation of enzyme activity involves the ensemble averaging of many cells, loss of spatial relationships and is often biased to abundant proteins. Here the authors develop activity-dependent proximity ligation to quantify enzyme activity at the cellular and sub-cellular level in relevant biological contexts.

    • Gang Li
    • , Jeffrey E. Montgomery
    • , Mark A. Eckert
    • , Jae Won Chang
    • , Samantha M. Tienda
    • , Ernst Lengyel
    •  & Raymond E. Moellering
  • Article
    | Open Access

    Extracellular vesicles (EV)-associated RNAs are serum biomarkers potentially exploitable for early cancer diagnosis. Here the authors develop a catalyzed hairpin DNA circuit within a cationic lipid-polymer hybrid nanoparticle that can detect low–level EV-associated RNAs in early stage cancer patients.

    • Jiaming Hu
    • , Yan Sheng
    • , Kwang Joo Kwak
    • , Junfeng Shi
    • , Bohao Yu
    •  & L. James Lee
  • Article
    | Open Access

    The lack of models that mimic hepatitis B virus (HBV) infection in a physiologically relevant context has hampered drug development. Here, Winer et al. establish a self-assembling, primary hepatocyte co-culture system that can be infected with patient-derived HBV without further modifications.

    • Benjamin Y. Winer
    • , Tiffany S. Huang
    • , Eitan Pludwinski
    • , Brigitte Heller
    • , Felix Wojcik
    • , Gabriel E. Lipkowitz
    • , Amit Parekh
    • , Cheul Cho
    • , Anil Shrirao
    • , Tom W. Muir
    • , Eric Novik
    •  & Alexander Ploss
  • Article
    | Open Access

    The guanine nucleotide exchange factor SOS is a critical intermediary that transduces receptor tyrosine kinase stimulation into Ras activation. Here the authors develop a single molecule assay in which SOS is captured from raw lysates using Ras-functionalized membrane microarrays and uncover an autoinhibitory mechanism of SOS regulation.

    • Young Kwang Lee
    • , Shalini T. Low-Nam
    • , Jean K. Chung
    • , Scott D. Hansen
    • , Hiu Yue Monatrice Lam
    • , Steven Alvarez
    •  & Jay T. Groves
  • Article
    | Open Access

    Monitoring DNA binding and single-base mismatches accurately in real time is difficult, especially for miniaturized devices. Here the authors report a graphene field-effect transistor array capable of reliably measuring DNA hybridization kinetics and affinity at the picomolar level.

    • Shicai Xu
    • , Jian Zhan
    • , Baoyuan Man
    • , Shouzhen Jiang
    • , Weiwei Yue
    • , Shoubao Gao
    • , Chengang Guo
    • , Hanping Liu
    • , Zhenhua Li
    • , Jihua Wang
    •  & Yaoqi Zhou
  • Article
    | Open Access

    Single cells can display large heterogeneity in gene induction. Here, Aymoz et al. present an expression reporter based on protein translocation that can accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution of less than one minute.

    • Delphine Aymoz
    • , Victoria Wosika
    • , Eric Durandau
    •  & Serge Pelet
  • Article
    | Open Access

    Progress in drug discovery can be hampered by a limited exploration of chemical space and the difficulty in assessing the full range of drug candidates’ effects on living cells. Here the authors describe a cell-based assay to distinguish between off-target and specific effects of candidate compounds targeting micro RNAs.

    • Benjamin Haefliger
    • , Laura Prochazka
    • , Bartolomeo Angelici
    •  & Yaakov Benenson
  • Article
    | Open Access

    Culturing normal primary breast cells that express the oestrogen receptor is difficult. Here, the authors isolate oestrogen receptor positive normal breast cells using the cell surface markers CD166 and CD117, and show that the cultures can be repeatedly passaged and retain oestrogen receptor protein expression.

    • Agla J. Fridriksdottir
    • , Jiyoung Kim
    • , René Villadsen
    • , Marie Christine Klitgaard
    • , Branden M. Hopkinson
    • , Ole William Petersen
    •  & Lone Rønnov-Jessen
  • Article |

    Bioassays are the standard way to measure prion infectivity titres, but can be time-consuming. In this study, bioassays are compared with a modified version of the protein misfolding cyclic amplification technique with beads (PMCAb), demonstrating that PMCAb can be more precise and faster than bioassays.

    • Natallia Makarava
    • , Regina Savtchenko
    • , Irina Alexeeva
    • , Robert G. Rohwer
    •  & Ilia V. Baskakov
  • Article |

    Protein interactions in biological environments are expected to differ from the situationin vitro. In this study, a thermophoresis-based technique is described that allows the analysis of protein and small-molecule interactions in biological liquids; the work may allow more efficient drug development.

    • Christoph J. Wienken
    • , Philipp Baaske
    • , Ulrich Rothbauer
    • , Dieter Braun
    •  & Stefan Duhr