Table of contents

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Protocol Update

SIFT missense predictions for genomes pp1 - 9

Robert Vaser, Swarnaseetha Adusumalli, Sim Ngak Leng, Mile Sikic & Pauline C Ng


This is an update to the SIFT protocol published in 2009, which uses SIFT 4G to provide SIFT scores from the genomes of more than 200 organisms.



Accelerated gas-liquid visible light photoredox catalysis with continuous-flow photochemical microreactors pp10 - 21

Natan J W Straathof, Yuanhai Su, Volker Hessel & Timothy Noël


In this protocol, the construction and use of an operationally simple photochemical microreactor for visible light gas-liquid photoredox catalysis is described. The procedure includes details on how to set up and use the microreactor.

Antibody-coupled siRNA as an efficient method for in vivo mRNA knockdown pp22 - 36

Nicole Bäumer, Neele Appel, Lisa Terheyden, Frank Buchholz, Claudia Rossig, Carsten Müller-Tidow, Wolfgang E Berdel & Sebastian Bäumer


This virus-free approach for mRNA knockdown in vivo uses the siRNA carrier peptide protamine, chemically coupled to a cell surface receptor internalizing antibodies via sulfo-SMCC, to provide protection and guidance for the targeted application of siRNA.

Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis pp37 - 45

Mostafa Zarei, Adrian Sprenger, Michal Rackiewicz & Joern Dengjel


This protocol describes phosphopeptide fractionation for LC-MS/MS analysis using a dual ERLIC-SCX solid-phase extraction method.

Enhancer scanning to locate regulatory regions in genomic loci pp46 - 60

Melissa Buckley, Anxhela Gjyshi, Gustavo Mendoza-Fandiño, Rebekah Baskin, Renato S Carvalho, Marcelo A Carvalho, Nicholas T Woods & Alvaro N A Monteiro


This protocol for enhancer scanning to locate regulatory regions in genomic loci enables the identification of candidate functional SNPs within a locus during functional analysis of genome-wide association studies.

Comprehensive multilevel in vivo and in vitro analysis of heart rate fluctuations in mice by ECG telemetry and electrophysiology pp61 - 86

Stefanie Fenske, Rasmus Pröbstle, Franziska Auer, Sami Hassan, Vanessa Marks, Danius H Pauza, Martin Biel & Christian Wahl-Schott


This protocol describes assays for assessing heart rate fluctuations in mice or tissue isolated from mice, including by using in vivo telemetry and in vitro electrophysiology of intact sinoatrial network preparations or isolated single sinoatrial node cells.

Studying extracellular vesicle transfer by a Cre-loxP method pp87 - 101

Anoek Zomer, Sander Christiaan Steenbeek, Carrie Maynard & Jacco van Rheenen


This protocol describes a Cre-loxP method to measure uptake of extracellular vesicles (EVs). Uptake of EVs released from cells that express Cre recombinase is detected in reporter cell lines as a switch from DsRed to eGFP expression.

Integrated analysis of shotgun proteomic data with PatternLab for proteomics 4.0 pp102 - 117

Paulo C Carvalho, Diogo B Lima, Felipe V Leprevost, Marlon D M Santos, Juliana S G Fischer, Priscila F Aquino, James J Moresco, John R Yates III & Valmir C Barbosa


This protocol describes PatternLab 4.0, an integrated software environment for the analysis of shotgun proteomics data.

MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems pp118 - 133

Tetsushi Sakuma, Shota Nakade, Yuto Sakane, Ken-Ichi T Suzuki & Takashi Yamamoto


This Protocol describes the CRIS-PITCh and TAL-PITCh systems for MMEJ-based gene targeting using CRISPR-Cas or TALENs. The approach may be particularly useful in systems where HR- or NHEJ-mediated targeting is inefficient.

Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics pp134 - 148

Majid Ebrahimi Warkiani, Bee Luan Khoo, Lidan Wu, Andy Kah Ping Tay, Ali Asgar S Bhagat, Jongyoon Han & Chwee Teck Lim


Viable circulating tumor cells are isolated using easily fabricated microfluidic devices. The hydrodynamic forces present in curvilinear microfluidic channels separate cells on the basis of size.

Scalable synthesis and post-modification of a mesoporous metal-organic framework called NU-1000 pp149 - 162

Timothy C Wang, Nicolaas A Vermeulen, In Soo Kim, Alex B F Martinson, J Fraser Stoddart, Joseph T Hupp & Omar K Farha


Metal-organic frameworks (MOFs) are porous, crystalline materials with well-defined structures that can be used in many applications, from gas storage to catalysis and drug storage. This protocol is for the preparation of the MOF NU-1000.

Correlated confocal and super-resolution imaging by VividSTORM pp163 - 183

László Barna, Barna Dudok, Vivien Miczán, András Horváth, Zsófia I László & István Katona


In this Protocol, Barna et al. describe how to use VividSTORM software to correlate the cellular information obtained from confocal imaging with single-molecule localization information obtained from superresolution STORM imaging.

Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes pp184 - 191

Nimali P Withana, Megan Garland, Martijn Verdoes, Leslie O Ofori, Ehud Segal & Matthew Bogyo


This protocol describes the use of fluorescently quenched activity-based probes (qABPs) that target cysteine cathepsins. This approach allows for tracking of protease activity in tissue samples, which can be used for the detection of tumor resection margins.



Corrigendum: Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts p192

Francois-Xavier Theillet, Honor May Rose, Stamatios Liokatis, Andres Binolfi, Rossukon Thongwichian, Marchel Stuiver & Philipp Selenko


Corrigendum: Cobalt-based nanocatalysts for green oxidation and hydrogenation processes p192

Rajenahally V Jagadeesh, Tobias Stemmler, Annette-Enrica Surkus, Matthias Bauer, Marga-Martina Pohl, Jörg Radnik, Kathrin Junge, Henrik Junge, Angelika Brückner & Matthias Beller


Corrigendum: The isolation and culture of endothelial colony-forming cells from human and rat lungs p192

Rajesh S Alphonse, Arul Vadivel, Shumei Zhong, Suzanne McConaghy, Robin Ohls, Mervin C Yoder & Bernard Thébaud



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