The voltage-dependent anion channel (VDAC) is an abundant and multifunctional outer mitochondrial membrane protein, playing key roles in neurodegeneration, apoptosis, and mitochondrial membrane biogenesis. Here, we present a protocol to produce and reconstitute high yields of detergent-solubilized VDAC, expressed as inclusion bodies in E. coli. We describe steps for purification by affinity chromatography and refolding in lauryldimethylamine-N-oxide (LDAO). We then detail procedures for reconstituting VDAC into membrane vesicles to assay its channel and phospholipid scramblase activity via fluorescence-based assays. For complete details on the use and execution of this protocol, please refer to Bergdoll et al.,1 Queralt-Martín et al., 2 and Jahn et al.3.

Selenoprotein thioredoxin reductase 1 (TXNRD1) is a promising therapeutic target, with several inhibitors reported to inhibit TXNRD1 activity. These inhibitors have the potential for applications such as anti-tumor medications. Here, we present a protocol for assessing irreversible inhibitors of TXNRD1. We describe four assays covering cellular TXNRD activity measurement, recombinant enzyme-based activity determination, differential scanning fluorimetry (DSF), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This protocol will facilitate the screening and development of potential small-molecule inhibitors of TXNRD1.

Engagement of TRAIL or Fas death receptors can trigger the assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) signaling complexes that promote nuclear factor κB (NF-κB) activation. Here, we present a protocol for immunoprecipitation of TRAIL- or Fas-induced FADDosomes from human cell lines. We describe steps for stimulating human cells with TRAIL or Fas ligand, followed by preparation of membrane death receptor-associated, as well as cytoplasmic FADDosome, signaling complexes. This protocol has application in the analysis of death receptor-induced signaling complex formation. For complete details on the use and execution of this protocol, please refer to Davidovich et al.1.

The aggregation and spreading of \"tau-seeds\" are key for the development and progression of tauopathies, including Alzheimer\'s disease. Here we describe the steps to isolate and analyze biochemically active tau-seeds from human, mouse, and cell origin. We detail the procedure to isolate soluble tau-seeds by size exclusion chromatography and seeding assay. The isolated tau-seed can be further analyzed to determine the interactome by mass spectrometry. This workflow identifies protein-protein interactors of tau-seeds, providing a useful tool for finding new therapeutic targets. For complete details on the use and execution of this protocol, please refer to Martinez et al.1.

AS1411-NCL-MDM2-based proteolysis-targeting chimeras (ANM-PROTACs) are capable of inducing selective degradation of transcription factors (TFs) in tumor cells. Here, we present a protocol for constructing ANM-PROTACs. We describe steps for molecular design of the ANM-PROTACs, assembly and characterization of the ANM-PROTACs, and initial assessment of in vitro TF degradation potency. We then detail procedures for validation of selective degradation of TFs via proteomic analysis. This protocol has been successfully applied to degrade various TFs across multiple tumor cell lines. For complete details on the use and execution of this protocol, please refer to Fu et al.1.

Literature reports suggest that the presence of proteins in pomegranate seeds is responsible for sensitization and IgE-mediated allergic reactions. The objective of this study was the analysis of a pomegranate seed extract and the isolation and characterization of proteins contained in high amounts. The extract characterization showed a protein profile with main bands at about 18 kDa and below 10 kDa upon SDS-PAGE, and molecules were recognized by specific IgEs upon immunoblotting. Then, two new 2S albumins, a monomeric and a heterodimeric one, were isolated by using classical biochemical methods. They were identified via direct protein sequencing and mass spectrometry, and their primary structure was analyzed and compared with homologous allergenic proteins via bioinformatics. In an Italian population of 703 suspected allergic patients, analyzed by using the FABER® test, the frequency of sensitization to the monomeric and heterodimeric 2S albumins was 1.7% and 0.28%, respectively. This study reports for the first time the isolation and characterization of two 2S albumins from pomegranate seeds. The clinical relevance of these molecules needs further investigation, for instance in populations having different exposures and allergy profiles.

Here, we present a protocol to quantify interactions among difficult-to-express proteins from Drosophila cells using the select western blot-free tagged-protein interaction (SWFTI) assay. We describe steps for plasmid design, cell plating, protein expression, and immunoprecipitation preparation. We then detail procedures for protein labeling, gel purification, and protein quantification. This protocol offers a fluorescence-based technique for rapid quantification of ectopically expressed proteins that are fused to SNAP and CLIP tags without the need for membrane transfer. For complete details on the use and execution of this protocol, please refer to Lin et al.1.

Brown adipose tissue (BAT) is mitochondria rich, enabling high oxidative metabolism for non-shivering thermogenesis. The release of large/small extracellular vesicles (EVs) containing mitochondria or mitochondrial fragments, termed mito-EVs, may support mitochondrial quality control or intercellular communication. We present a protocol to isolate and characterize mito-EVs. We detail steps for BAT processing, cell debris removal, differential centrifugation (dC), and mito-EV analysis by flow cytometry and immunoblotting assays. For complete details on the use and execution of this protocol, please refer to Rosina et al.1.

Protein-nucleic acid interactions drive some of the most important physiological events in cells. Here, we present a protocol for detecting protein-DNA or protein-RNA interactions in vitro. We describe steps for labeling nucleic acid species and electrophoretic mobility shift assays (EMSAs). This protocol can be used to confirm suspected in vivo interactions using recombinantly expressed/purified proteins of interest and a nucleic acid substrate. It can further be used to investigate mutations that can disrupt interaction or compensatory mutations that restore it. For complete details on the use and execution of this protocol, please refer to Mansouri-Noori et al.1.

Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting. For complete details on the use and execution of this protocol, please refer to Maghe et al.1.