Cancer Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 690 Views Oct 5, 2022

RNA binding proteins (RBPs) are critical regulators of cellular phenotypes, and dysregulated RBP expression is implicated in various diseases including cancer. A single RBP can bind to and regulate the expression of many RNA molecules via a variety of mechanisms, including translational suppression, prevention of RNA degradation, and alteration in subcellular localization. To elucidate the role of a specific RBP within a given cellular context, it is essential to first identify the group of RNA molecules to which it binds. This has traditionally been achieved using cross-linking-based assays in which cells are first exposed to agents that cross-link RBPs to nucleic acids and then lysed to extract and purify the RBP-nucleic acid complexes. The nucleic acids within the mixture are then released and analyzed via conventional means (e.g., microarray analysis, qRT-PCR, RNA sequencing, or Northern blot). While cross-linking-based ribonucleoprotein immunoprecipitation (RIP) has proven its utility within some contexts, it is technically challenging, inefficient, and suboptimal given the amount of time and resources (e.g., cells and antibodies) required. Additionally, these types of studies often require the use of over-expressed versions of proteins, which can introduce artifacts. Here, we describe a streamlined version of RIP that utilizes exclusion-based purification technologies. This approach requires significantly less starting material and resources compared to traditional RIP approaches, takes less time, which is tantamount given the labile nature of RNA, and can be used with endogenously expressed proteins. The method described here can be used to study RNA-protein interactions in a variety of cellular contexts.


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0 Q&A 4617 Views Jun 5, 2018
The glycosaminoglycan hyaluronan (HA) is a key component of the extracellular matrix. The molecular weight of HA is heterogeneous and can reach from several million to several hundred daltons. The effect of HA on cell behavior is size dependent; fragmented HA acts as a danger signal, stimulates cell migration and proliferation and is proinflammatory, native high molecular weight HA suppresses inflammation. Therefore, it is important to analyze HA size distribution when studying the role of HA in tissue homeostasis and pathology. This protocol describes isolation of HA from mouse mammary glands but can also be applied to other tissues. The quality of the isolated HA is sufficient to analyze size distribution by gel electrophoresis (Calabro et al., 2000).
0 Q&A 17519 Views Mar 5, 2017
Phosphoinositides are rare membrane lipids involved in the control of the major cellular functions and signaling pathways. They are able to recruit specific effector proteins to the cytosolic face of plasma membrane and organelles to coordinate a vast variety of signaling and trafficking processes, as well to maintain specific identity of the different subcellular compartments (Di Paolo and De Camilli, 2006; Lemmon, 2003). Therefore, analysis of these effectors’ binding properties and specificity towards different phosphoinositides is crucial for the understanding of their cellular functions. This protocol describes a method to characterize the binding of proteins to different phosphoinositide-containing vesicles.
0 Q&A 13533 Views Jan 20, 2016
This is an assay designed to examine the radioactive phosphorous incorporation when the molecule is being synthesized, which means that only de novo synthesized phospholipids can be detected. Thus, with this technique it is possible to detect in vitro phospholipid synthesis under different required experimental conditions respect to controls (Guido and Caputto, 1990; Ferrero et al., 2014). There are different types of lipids. Among them we can find phospholipids, which contain glycerol esterified with two fatty acyl chains and a phosphate group that can also be bound to an organic molecule that acts as “hydrophilic head”, as shown in Figure 1 for the case of phosphatidylcholine. This structure confers amphipathic properties to lipid molecules that allow them to form lipid bilayers, making phospholipids the main components of biological membranes.


Figure 1. Representation of phospholipid structure. Extracted from: http://bio1151.nicerweb.com/Locked/media/ch05/phospholipid.html



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