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...
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 ...
Hou, Yingjie Lu, Heng Sun, Le Zhang, Yaoyang Jiang, Hong
Published in
STAR protocols
Mammalian cyclic dinucleotide 2'3'-cGAMP functions as a second messenger in innate immune response. Here, we report a protocol to utilize 2'3'-cGAMP photoaffinity probes to capture 2'3'-cGAMP-binding or 2'3'-cGAMP-interacting proteins from HeLa cell lysate for in-gel visualization by fluorescent imaging or identification by SILAC-based quantitative...
Yan, Zhenzhen Liu, Hansen Gao, Chengjiang
Published in
STAR protocols
Upon viral infection, several proteins in the innate signaling pathway form aggregates, which in turn promote the activation of innate antiviral immune response. In this protocol, we use herpes simplex virus type 1 (HSV-1) to infect mouse peritoneal macrophages, and show how to detect the aggregation of TBK1 upon viral infection. The protocol is ad...
Nahlé, Sarah Quirion, Laura Boulais, Jonathan Bagci, Halil Faubert, Denis Gingras, Anne-Claude Côté, Jean-François
Published in
STAR protocols
Proximity-dependent biotinylation (BioID) screens are excellent tools to capture in cellulo interactomes for a large variety of baits, including transient and weak affinity interactions, as well as localization-specific proximity components, which are much harder to detect with conventional approaches. Here, we describe the major starting steps and...
Yang, Xiaoqi Fox, Alisa Powell, Rebecca L R
Published in
STAR protocols
Antibodies in milk obtained from those previously SARS-CoV-2-infected or vaccinated against COVID-19 may provide passive immunity to the breastfed infant. Few assays have been established to measure antibodies in human milk, despite the public health importance of this topic. In the present protocol, we describe an optimized indirect ELISA assay ai...
Zhou, Junzhi Liang, Qian Dong, Maogong Ma, Xiaohe Jin, Yaqi Guan, Di Liu, Jiang Wang, Miao Cong, Yu-Sheng
Published in
STAR protocols
Ubiquitin-fold modifier 1 (UFM1) system is a recently identified ubiquitin-like modification with essential biological functions. Similar to ubiquitination, the covalent conjugation of UFM1 (UFMylation) to target proteins involves a three-step enzymatic cascade catalyzed sequentially by UFM1-activating enzyme 5 (UBA5, E1), UFM1-conjugating enzyme 1...
Kim, Kijun Kim, V Narry
Published in
STAR protocols
We describe a protocol to conduct a high-throughput in vitro processing assay, using 1,881 human primary microRNAs (pri-miRNAs) and recombinant Microprocessor complex, followed by deep sequencing library generation. This comprehensive approach allows the mapping of cleavage sites and the measurement of processing efficiency of a large number of sub...
Zupancic, Jennifer M. Desai, Alec A. Tessier, Peter M.
Published in
STAR Protocols
The generation of high-affinity nanobodies for diverse biomedical applications typically requires immunization or affinity maturation. Here, we report a simple protocol using complementarity-determining region (CDR)-swapping mutagenesis to isolate high-affinity nanobodies from common framework libraries. This approach involves shuffling the CDRs of...
Gopalan, Sneha Fazzio, Thomas G.
Published in
STAR Protocols
Genome-wide chromatin mapping approaches typically focus on one protein at a time. We recently developed multi-CUT&Tag, which enables simultaneous mapping of multiple chromatin proteins in the same single cells or pools of cells. Using barcoded adapters loaded onto antibody-protein A-Tn5 transposase complexes, multi-CUT&Tag marks the locations of e...
