Bartuli, Julia Lorenzi, Isotta Backes, Simone Grimm, Clemens Fischer, Utz
Published in
STAR protocols
The functional and structural characterization of macromolecular complexes requires protocols for their native isolation. Here, we describe a protocol for this task based on the recombinant poxvirus Vaccinia expressing tagged proteins of interest in infected cells. Tagged proteins and their interactors can then be isolated via affinity chromatograp...
Kanemaru, Hisashi Mizukami, Yukari Kaneko, Akira Kajihara, Ikko Fukushima, Satoshi
Published in
STAR protocols
Current standard assays to analyze lymphocyte-mediated antitumor cytotoxicity employ radioisotopic or fluorescent labels. However, such assays are not suitable for real-time analysis. Here we describe a protocol that facilitates the analysis of lymphocyte-mediated toxicity using a label-free, impedance-based real-time cell analyzer. This analyzer m...
Wang, Qi Tang, Bin Tang, Jianrong
Published in
STAR protocols
Deep brain stimulation (DBS) entails neurosurgery to implant electrodes in specific brain structures to modulate the behavior of a particular neural circuit. DBS is best known for treating advanced Parkinson disease and can potentially be applicable to other motor and even cognitive dysfunctions. Here, we describe a detailed protocol allowing for e...
Bou-Nader, Charles Zhang, Jinwei
Published in
STAR protocols
Host tRNAs specifically interact with the matrix domain (MA) of HIV-1 major structural polyprotein, Gag, to control its membrane localization and virion assembly. In this protocol, we describe the purification and engineering of HIV-1 MA and tRNA, and the co-crystallization and structure determination of the complex using X-ray crystallography. Rat...
Dong, Shupeng Xie, Xiaochun Chen, Yujie Long, Gang Liang, Qiming Zhao, Zhen Zeng, Jianxiong
Published in
STAR protocols
Zika virus (ZIKV) is linked to congenital defects including microcephaly. An infection model that can recapitulate most microcephaly-related phenotypes is crucial for understanding ZIKV pathogenesis. Here, we present a protocol to generate ZIKV from an infectious clone through a reverse genetic system and subsequently perform embryonic brain infect...
Zhang, Jin-Fan Mehta, Sohum Zhang, Jin
Published in
STAR protocols
Fluorescent protein (FP)-based kinase activity biosensors are powerful tools for probing the spatiotemporal dynamics of signaling pathways in living cells. Yet, the limited sensitivity of most kinase biosensors restricts their reliable application in high-throughput detection modalities. Here, we report a protocol for using an ultrasensitive excita...
Lee, Seongsin Kim, Yumi
Published in
STAR protocols
Ribosomes are composed of a mix of ribosomal RNAs and proteins; this composition varies depending on time, condition, and organism. Here, we present an optimized protocol for preparation of intact ribosomes from the skeletal muscle of the turquoise killifish. We also detail the steps for ribosome quantification and cryo-EM grid preparation. This pr...
Marquez-Galera, Angel de la Prida, Liset M Lopez-Atalaya, Jose P
Published in
STAR protocols
Bulk-tissue RNA-seq is widely used to dissect variation in gene expression levels across tissues and under different experimental conditions. Here, we introduce a protocol that leverages existing single-cell expression data to deconvolve patterns of cell-type-specific gene expression in differentially expressed gene lists from highly heterogeneous ...
Rabenius, Adelina Chandrakumaran, Sajitha Sistonen, Lea Vihervaara, Anniina
Published in
STAR protocols
Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide analytical steps to (1) identify transcribed regulatory elements de novo genome-wide, (2) quantify engaged transcription complexes at enhancers, promoter-pr...
Desch, Kristina Schuman, Erin M Langer, Julian D
Published in
STAR protocols
Cellular processes require tight and coordinated control of protein abundance, localization, and activity. One of the core mechanisms to achieve specific regulation of proteins is protein phosphorylation. Here we present a workflow to monitor protein abundance and phosphorylation in primary cultured neurons using liquid chromatography-coupled mass ...