Molecular Biology


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0 Q&A 305 Views Nov 20, 2023

The lipid bilayers of the cell are composed of various lipid classes and species. These engage in cell signaling and regulation by recruiting cytosolic proteins to the membrane and interacting with membrane-embedded proteins to alternate their activity and stability. Like lipids, membrane proteins are amphipathic and are stabilized by the hydrophobic forces of the lipid bilayer. Membrane protein–lipid interactions are difficult to investigate since membrane proteins need to be reconstituted in a lipid-mimicking environment. A common and well-established approach is the detergent-based solubilization of the membrane proteins in detergent micelles. Nowadays, nanodiscs and liposomes are used to mimic the lipid bilayer and enable the work with membrane proteins in a more natural environment. However, these protocols need optimization and are labor intensive. The present protocol describes straightforward instructions on how the preparation of lipids is performed and how the lipid detergent mixture is integrated with the membrane protein MARCH5. The lipidation protocol was performed prior to an activity assay specific to membrane-bound E3 ubiquitin ligases and a stability assay that could be used for any membrane protein of choice.

0 Q&A 4535 Views Apr 20, 2020
Expression levels of cellular proteins can be affected by various perturbations, such as genetic knockout of interactors, drug treatments or cell stress. To specifically measure the effects on protein levels post-synthesis under different experimental conditions, it is important to compensate for transcriptional and other upstream changes. Here, we provide a protocol for a dual-fluorescence flowcytometry-based assay to determine protein levels. The protein of interest is genetically linked to enhanced GFP (eGFP) followed by a viral 2A self-cleaving peptide sequence and mCherry. As a result, translation of the reporter construct leads to two fluorescent protein products from the same mRNA template, which enables unambiguous protein expression analysis with normalization across samples.
0 Q&A 4854 Views Sep 20, 2018
Structural stability of the capsid core is a critical parameter for the productive infection of a cell by a retrovirus. Compromised stability can lead to premature core disassembly, exposure of replication intermediates to cytosolic nucleic acid sensors that can trigger innate antiviral responses, and failure to integrate the proviral genome into the host DNA. Thus, core stability is a critical feature of viral replicative fitness. While there are several well-described techniques to assess viral capsid core stability, most are generally time and labor intensive. Recently, our group compared the relative stability of murine leukemia virus capsid cores using an in vitro detergent-based approach combined with ultracentrifugation against the popular fate of capsid assay. We found that both methods reached similar conclusions, albeit the first method was a significantly simpler and faster way to assess relative capsid core stability when comparing viral mutants exhibiting differences in core stability.
0 Q&A 10474 Views Aug 20, 2017
Pulse-chase technique is a method widely used to assess protein or mRNA stability. The principle of pulse-chase relies on labeling proteins or mRNA produced during a short period of time called ‘pulse’ and then following the rate of disappearance of those labeled proteins over a period of time called ‘chase’. This technique thus allows quantitative analysis of modulation of protein or mRNA stability under different treatments or culturing conditions.



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