Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host\'s immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

Natural products, while valuable for drug discovery, encounter limitations like uncertainty in targets and toxicity. As an important active ingredient in traditional Chinese medicine, celastrol exhibits a wide range of biological activities, yet its mechanism remains unclear. In this study, they introduced an innovative \"Degradation-based protein profiling (DBPP)\" strategy, which combined PROteolysis TArgeting Chimeras (PROTAC) technology with quantitative proteomics and Immunoprecipitation-Mass Spectrometry (IP-MS) techniques, to identify multiple targets of natural products using a toolbox of degraders. Taking celastrol as an example, they successfully identified its known targets, including inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3Kα), and cellular inhibitor of PP2A (CIP2A), as well as potential new targets such as checkpoint kinase 1 (CHK1), O-GlcNAcase (OGA), and DNA excision repair protein ERCC-6-like (ERCC6L). Furthermore, the first glycosidase degrader is developed in this work. Finally, by employing a mixed PROTAC toolbox in quantitative proteomics, they also achieved multi-target identification of celastrol, significantly reducing costs while improving efficiency. Taken together, they believe that the DBPP strategy can complement existing target identification strategies, thereby facilitating the rapid advancement of the pharmaceutical field.

Connexins (Cxs) are transmembrane proteins which form hemichannels and gap junction channels at the plasma membrane. These channels allow the exchange of ions and molecules between the intra- and extracellular space and between cytoplasm of adjacent cells, respectively. The channel function of Cx assemblies has been extensively studied; however, \"noncanonical\" functions have emerged in the last few decades and have capture the attentions of many researchers, including the role of some Cxs as gene modulators or transcription factors. In this chapter, we describe a protocol to study the interaction of Cx46 with DNA in HeLa cells. These methods can facilitate understanding the role of Cxs in physiological processes and pathological mechanisms, including, for example, the contribution of Cx46 in maintaining stemness of glioma cancer stem cells.

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and transcriptional regulation. Therefore, understanding PPIs is an important starting point for further investigation of the function of the target protein. In this study, we propose a simple method to determine the binding of two target proteins by introducing mammalian expression vectors into HEK-293 cells using the polyethylenimine method, lysing the cells in homemade protein lysis buffer, and pulling down the target proteins on an epitope tag affinity gel. In addition, the PPI between the various epitope tag fused proteins can be confirmed by using affinity antibodies against each tag instead of the epitope tag affinity gel. This protocol could also be used to verify various PPIs, including nuclear extracts, from other cell lines. Therefore, it can be used as a basic method in a variety of PPI experiments. Proteins degrade by extended time course and repeated freeze-thaw cycles. Therefore, cell lysis, immunoprecipitation, and immunoblotting should be performed as seamlessly as possible.

The quantitative and qualitative biochemical description of molecular interactions is fundamental to the study of ligand/receptor pairs and their structure/function relationships. Bioactive peptides often are active at (sub-)nanomolar concentrations, indicating they have a high affinity for their sites of action, notably binding sites on receptors. Since such receptor proteins are commonly of low abundance, highly sensitive detection methods are required to study these ligand/receptor interactions. We present a protocol for an inexpensive luminescence-based detection setup in which the peptide ligand of interest is extended with the 11-amino acid HiBiT tag. This tag can be quantified easily down to fmol amounts by its ability to reconstitute the enzymatic activity of LgBiT, a truncated version of the Oplophorus gracilirostris luciferase.

Affinity purification-Mass spectroscopy (AP-MS) is a biochemical technique to identify the novel protein-protein interaction that occurs in the most relevant physiological conditions, whereas co-immunoprecipitation (Co-IP) is used to study the interaction between two known protein partners that are expressed in the native physiological conditions. Both AP-MS and Co-IP techniques are based on the ability of the interacting partners to pull-down with protein of interest. In this chapter, we have explained the AP-MS and Co-IP methods to study protein-protein interactions in the plant cells.

The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as a powerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don\'ts will enable beginners and experienced scientists to study the import and assembly of MRPs.

There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3\',4\':6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38.

The long non-coding RNAs (lncRNAs) other than rRNA and tRNA were earlier assumed to be \'junk genomic material\'. However, recent advancements in genomics methods have highlighted their roles not only in housekeeping but also in the progression of diseases like cancer as well as viral infections. lncRNAs owing to their length, have both short-range and long-range interactions resulting in complex folded structures that recruit various biomolecules enabling lncRNAs to undertake their various biological functions. Using cell lysate pull-down assays increasing number of lnRNAs-interacting proteins are being identified. These interactions can be further exploited to develop targeted novel therapeutic strategies to inhibit lncRNA-protein interactions. This review attempts to succinctly techniques that can identify and characterize the lnRNAs-protein interactions (i.e. affinity, stoichiometry, and thermodynamics). Furthermore, using other sophisticated biophysical techniques, one can also perform size estimations, and determine low-resolution structures. Since these methods study the biomolecules in solution, large-scale structural observations can be performed in real-time. This review attempts to briefly introduce the readers to biochemical and biophysical techniques, such that they can utilize these methods to obtain a holistic characterization of the biomolecules of interest. Additionally, it should be noted that the use of these methods is not limited to the characterization of the interacting molecules but can also be used to determine the efficacy of the therapeutic molecules to disrupt these interactions.

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein crosslinking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.