Max Planck Institute of Immunobiology and Epigenetics

4 ARTICLES PUBLISHED IN JoVE

Immunology and Infection

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Jordan F. Hastings¹, Jeremy Z.R. Han¹, Robert F. Shearer^1,2, Sean P. Kennedy^1,3, Mary Iconomou^1,4, Darren N. Saunders⁵, David R. Croucher^1,6,7

¹The Kinghorn Cancer Centre, Garvan Institute of Medical Research, ²Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, ³RCSI Molecular Medicine, Royal College of Surgeons in Ireland, ⁴Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, ⁵School of Medical Sciences, University of New South Wales, ⁶St Vincent's Hospital Clinical School, University of New South Wales, ⁷School of Medicine and Medical Science, University College Dublin

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Biology

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Visnja Jevtic¹, Petra Kindle¹, Sergiy V. Avilov¹

¹Imaging Facility, Max Planck Institute of Immunobiology and Epigenetics

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Genetics

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Chih-Hsiang Yang^1,2, John Longinotto², Ilaria Panzeri^1,2, Laura Arrigoni², Vanessa Wegert¹, Steffen Heyne², Gabriel Seifert³, Adelheid Lempradl^1,2, Ulrike Bönisch², John Andrew Pospisilik^1,2

¹Van Andel Institute, ²Max Planck Institute of Immunobiology and Epigenetics, ³Department of General and Visceral Surgery, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Immunology and Infection

Targeted Metabolomics on Rare Primary Cells

Katharina M. Glaser^*1, Mirijam Egg^*1, Sebastian Hobitz¹, Michael Mitterer¹, Dominik Schain-Zota¹, Katharina Schönberger¹, Konrad Schuldes¹, Nina Cabezas-Wallscheid¹, Tim Lämmermann¹, Angelika Rambold¹, Joerg M. Buescher¹

¹Max Planck Institute of Immunobiology and Epigenetics

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.