The detection of molecular events in living cells is booming. In this post, we look at 3 fluorescent probes that will undoubtedly count in the live-cell imaging landscape in 2017.
Today, I’d like to give you an overview about methods in actin research with validated R&D products and kits which will allow you to measure binding to actin and effects on the polymerisation dynamics of actin.
Actin functions as one of the major cytoskeleton structures. It is involved in a plethora of processes in cell biology: stabilizing the cell shape, cell movements (e.g. cell migration) and intracellular movements and transport mechanisms.
Actin is a 43 kDa protein that is very highly conserved between species. Actin has three main isotypes (α-actin, β-actin and γ-actin), which show >90% amino-acid (aa) homology between isotypes and >98% homology within members of a particular isotypic group. [Read more…]
The term Autophagy was introduced by Christian de Duve during the Ciba Foundation Symposium on Lysosomes – which was held in London in February 1963. In 1974 he was honoured with the Nobel price in Physiology or Medicine for his pioneering research about peroxisomes and lysosomes. In 2016, once more, a pioneer in the field of autophagy research won the Nobel price: Yoshinori Ohsumi, a Japanese researcher, whose findings “led to a new paradigm in our understanding of how the cell recycles its content”.
Autophagy (Autophagocytosis) describes the fundamental catabolic mechanism during which cells degrade dysfunctional and unnecessary cellular components. This process is driven by the action of lysosomes and promotes survival during starvation periods as the cellular energy level can thus be maintained. [Read more…]
Research tools aimed at exploring mitochondria are becoming more and more popular and cover multiple experimental applications. They go from primary antibodies validated for immuno-assays (see “10 Mitochondrial marker antibodies“) to fluorescent probes for mitochondria-related events monitor in living-cells (e.g. MitoPeDPP for live cell imaging, mitophagy or singlet Oxygen (1O2) detection…). Here I’d like to introduce 3 new molecules developed by Focus Biomolecules to further decipher mitochondrial biology.
Determining the linkage of polyubiquitin on target proteins is challenging. The traditional methods are either through Mass Spectrometry or immunoblot with linkage specific antibodies, which are cumbersome. LifeSensors has developed the UbiTest assay, a more definitive method for demonstrating the ubiquitylation linkage of a protein, which is to couple immunoprecipitation of polyubiquitylated protein with digestion by a linkage specific deubiquitylase prior to immunoblot analysis. An increased signal for the unmodified substrate or a decreased signal of polyubiquitylated substrate at high molecular weight after K48/K63 specific DUB treatment is a clear indication that the protein was K48/K63 ubiquitylated. [Read more…]
Are you working on autophagy,? Or on GPCR de-orphaning? On the MAP kinase pathway or cellular metabolism? Maybe on pathways related to cellular metabolism?
Several compounds are known as modulators of these pathways and research fields and can be used as useful tools to study and characterize specific steps. Nonetheless, it is quite time consuming and expensive to order all these chemicals from different sources. Targeted compound libraries represent a convenient alternative. [Read more…]
Small GTP-binding proteins such as RhoA, Rac1, and Cdc42 are involved in regulating cell signalling pathways and impact a wide range of cellular processes, functions, and morphology. They bind and hydrolyze GTP, thus being switched from the activated form to the inactivated form (Fig 1).
The most prominent family of small G proteins is represented by the Ras superfamily of proteins. The Rho subfamily belonging to this superfamily consists of proteins like RhoA, Rac1, and Cdd42. These proteins have been shown to be involved in the regulation of actin dynamics, thus playing a crucial role in processes like cell movement, intracellular transport, and organelle development. While RhoA affects actin stress fibers, Rac1 exhibits effects on lamellipodia and Cdc42 on filopodia. [Read more…]
The Silicon Rhodamine-like (SiR) technology has significantly contributed to the recent development of DNA and cytoskeletal analysis by live cell imaging.
In 2014, two new Silicon Rhodamine-like (SiR) fluorescent probes were released for studying actin & tubulin by live cell imaging. SiR-Actin and SiR-Tubulin are fluorescent probes compatible with most microscopes (including super-resolution settings) that directly stain actin & tubulin without the need to transfect cells with vectors expressing fluorescently labeled Actin or Tubulin. The two original dyes were successfully followed by a new SiR-DNA probe in order to visualize DNA in living cells.
The existing SiR stains have a λabs of 652 nm and a λem of 674 nm to be used with the Cy5 filter (Fig 1).
However, the continuously growing number of researchers using these stains asked us whether stains with different biophysical properties would be made available. In other words, they were asking “is there another colour to allow for double staining e.g. of Actin and Tubulin in living cells?”