Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, thus impeding progress in understading structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need of long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here, we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/SDS PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.

Subcellular fractionation is an introductory step in a variety of experimental approaches designed to study intracellular components, like membranes and organelle systems. Subcellular fractions enriched in membranes of the Golgi apparatus of mammalian cells have been isolated to address localization and activity of proteins, including enzymes, to study intracellular membrane transport mechanisms, and to reconstitute in vitro cellular processes associated with the Golgi apparatus. Here, I describe methods to purify Golgi membranes by subcellular fractionation, to assay nucleotide sulfate (PAPS) uptake into Golgi vesicles, and to measure sulfate incorporation into in vitro synthesized glycosaminoglycans.

Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. In this protocol, we describe a procedure for the isolation of 70S ribosomes from the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We have successfully used this protocol in our study of the cyanobacterial ribosomal-associated protein LrtA, which is a homologue of bacterial HPF (hibernation promoting factor) ( Galmozzi et al., 2016 ).

Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, impeding progress in understanding the structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need for long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.

Argonaute (AGO) proteins play a key role in RNA silencing mechanisms. RNA silencing affects both RNA degradation and translation. The characterization of translation-associated RNA silencing mechanisms and components often requires polysome isolation and analysis. In this chapter, we describe the identification of AGO1 association with polysomes through polysome fractionation on sucrose gradient, preparation of proteins by filtration and concentration, and immunoblotting.

Protein misfolding, aggregation, and accumulation are a common hallmark in various neurodegenerative diseases. Invariably, the process of protein aggregation is associated with both a loss of the normal biological function of the protein and a gain of toxic function that ultimately leads to cell death. The precise origin of protein cytotoxicity is presently unclear but the predominant theory posits that smaller oligomeric species are more toxic than larger aggregated forms. While there is still no consensus on this subject, this is a central question that needs to be addressed in order to enable the design of novel and more effective therapeutic strategies. Accordingly, the development and utilization of approaches that allow the biochemical characterization of the formed oligomeric species in a given cellular or animal model will enable the correlation with cytotoxicity and other parameters of interest.Here, we provide a detailed description of a low-cost protocol for the analysis of protein oligomeric species from both yeast and mammalian cell lines models, based on their separation according to sedimentation velocity using high-speed centrifugation in sucrose gradients. This approach is an adaptation of existing protocols that enabled us to overcome existing technical issues and obtain reliable results that are instrumental for the characterization of the types of protein aggregates formed by different proteins of interest in the context of neurodegenerative disorders.

In actively growing cells, the rate of translation initiation is relatively rapid. As a result, multiple ribosomes simultaneously engaged in translation become spaced along single mRNA molecules. These structures, termed polysomes, can be separated from ribosomal subunits and single ribosomes because they migrate faster through sucrose gradients (Noll, 2008). In fact, polysomes containing various numbers of ribosomes can be resolved from one another since each ribosome adds substantial mass. It is straightforward to prepare bacterial lysates and resolve all the ribosomal fractions using sucrose gradient sedimentation. The resulting polysome \'profile\' can provide a sort of snapshot of the translation activity in the cell. Polysome analysis has been used to study the effects of mutations and/or growth conditions on translation and to address whether particular cellular components are associated with the translational machinery (Powers and Noller, 1990; Gregory et al., 1994; Moine and Dahlberg, 1994; Firpo et al., 1996; Fredrick et al., 2000; Ataide et al., 2009; Melamed et al., 2009; Saini et al., 2009). In combination with other techniques, polysome analysis has been used to deduce rate constants for certain phases of translation (e.g., initiation, elongation, termination) (Arava et al., 2003; 2005). Finally, use of purified polysomes in biochemical experiments has been instrumental for the isolation and characterization of translation factors such as ribosome recycling factor (RRF) (Hirashima and Kaji, 1972; Fujiwara et al., 2001; Hirokawa et al., 2002; Ito et al., 2002). Here, we describe a simple and convenient method of preparing and analyzing polysomes from Escherichia coli, which should be generally applicable to many bacteria. We also discuss parameters that influence the ribosome density on mRNA, which should be kept in mind when polysome profiles are being interpreted.