In this protocol we describe how to perform a ratiometric three-state stable isotope labeling by amino acids in cell culture (SILAC) approach that allows to map an open cellular compartment like the yeast nucleus. In particular, we focus on the proteomic sample preparation and provide instructions...
Transfections were performed as described in the protocol. Briefly, cells were seeded to different plates, and the next day, the DNA and Lipofectamine were mixed and added to the cells. Forty hours later, expression was detected by western blot or immunofluorescence. For imaging experiments, ...
However, the poor membrane permeability of the biotin phenol substrate and the inhibitory effect of peroxide on the enzyme's activity has hampered proximity labeling in certain cell culture systems and tissues. Here, we describe an APEX2 protocol that uses alternative peroxide and biotin phenol ...
Here, we present a protocol for identifying interactors of RNA-binding proteins (RBPs) in living HeLa cells using the APEX2 fusion construct. We describe steps for generation of RBP-APEX2, proximity biotin labeling, and preparation of labeled molecules for mass spectrometry analysis. This protocol...
Proximity labelingLipid droplets (LDs) are ubiquitous lipid storage organelles composed of a neutral lipid core surrounded by a phospholipid monolayer that is decorated with integral and peripheral proteins. Accurate...doi:10.1007/978-1-4939-9537-0_5Kirill Bersuker...