Acting and reacting in life science biotechnologies
For more than 2 years now, the Silicon Rhodamine-like (SiR) technology has allowed the live cell imaging field with fluorescence microscopy to evolve significantly.
Fluorescent SiR-probes have appeared as the best alternative tools for studying Actin (SiR-actin), Microtubules (SiR-Tubulin), DNA (SiR-DNA) and now lysosome (SiR-Lysosome) for live cell imaging. Who better to show this? Well, here’s how other researchers have been using them to get optimal results. [Read more…]
Today, I’d like to give you an overview about methods in actin research with validated R&DÂ 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 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?”
In the autumn of 2014, we presented new stains to conduct live cell imaging of Actin and Tubulin, which I covered in my post 2 new Actin and Tubulin live-cell imaging stains – without transfection!
Fig 1: Live MCF10A cells spheroid stained with SiR-DNA and imaged by confocal microscopy. Courtesy of C. Conrad and K. Jechow (Heidelberg).
SiR-Actin and SiR-Tubulin exhibit a number of benefits which have since then be experienced by a huge number of labs all over the world:
Since then, Spirochrome has added another stain with the same benefits: SiR-DNA, a far-red, fluorogenic, cell permeable and highly specific probe for DNA (see Fig 1).
From June 27th to June 29th, the SLAS – High Content Screening Conference took place in Dresden/Germany. The conference focussed on new essays and novel microscopy techniques.
Spirochrome and tebu-bio were selected to present a poster about the features of SiR-stains, which are excellent tools for phenotypic screening and high content screening (HCS) approaches.
If you are interested in learning more about these exciting live cell imaging tools, don’t hesitate to download our joint poster:
Any questions or comments? Please feel free to contact me with the form below!
Interested in learning more about tools like this?2015 was marked by the increasing number of studies related to cellular cytoskeletal dynamics. In this context, it should be noted that the new generation of live cell-imaging SiR-Actin & SiR-Tubulin stains have been widely embraced by Life Scientists. Launched a few months ago, these fluorescent probes are now regularly cited in the “Material & Methods” section of numerous prestigious publications as indicated by Ali (see his the posts “Live cell imaging tool SiR-actin on JBC frontcover” and “User experience of SiR-Actin and SiR-Tubulin Live Cell Imaging“).
Nevertheless, cytoskeletal interactions are still widely studied in “fixed” cells. In the same manner as for live cell-imaging, these experimental assays require high quality reagents too. With the help of our friends from Cystoskeleton Inc., we have selected 9 premium research tools validated for actin visualization in PFA-fixed cells. [Read more…]
For one year now,  SiR-Actin and SiR-Tubulin, the second generation of tools to stain actin and tubulin in living cells, have been available on the market – and although it may sound like an overstatement, these stains have changed the world of Live Cell Imaging. [Read more…]
In previous posts,  I invited you to look at versatile methods to
Fig. 1: Double-helical structure of actin filaments (provided by Cytoskeleton Inc.)
Today, let’s focus on a method which enables researchers to measure the ratio between monomeric globular actin (G actin) and filamentous actin (F actin) in cells, thus giving a good insight in the status of actin dynamics.
Fig. 1: Double-helical structure of actin filaments (provided by Cytoskeleton Inc.)
Actin dynamics – i.e. polymerization of G-actin to form filamentous F-actin or de-polymerization of F-actin – is a fundamental process in cell biology, as it is the basis of cell movement (e.g. cell migration) and intracellular movements and transport mechanisms. Globular-actin (G-actin) readily polymerizes under physiological conditions to form filamentous actin (F-actin) with the concomitant hydrolysis of ATP. F-actin is a double-helical filament (Fig. 1). Actin can polymerize from both ends in vitro. However, the rate of polymerization is not equal. This results in an intrinsic polarity in the actin filament. It has therefore become the convention to term the rapidly polymerizing end the plus-end or barbed-end (+) while the slow growing end is called the minus-end or pointed-end (-).
In this post, I’d like to concentrate on a method to measure polymerization of actin in biochemical assays. It’s the third post in a series of actin related publications which started with Focus on Actin staining and visualization and Focus on Actin – Detection of actin binding and actin binding proteins. In an upcoming blog I’ll be focussing on G-F actin ratio detection in cells. [Read more…]
Actin can exist in two forms: Globular subunit (G-actin) and Filamentous polymer (F-actin). Both forms of actin interact with a plethora of proteins in the cell. To date there are over 50 distinct classes of Actin-Binding Proteins (ABPs), and the inventory is still far from complete. Actin Binding Proteins allow the actin cytoskeleton to respond rapidly to cellular and extracellular signals and are integral to cytoskeletal involvement in many cellular processes. These include cell shape and motility, muscle contraction, intracellular trafficking, cell pathogenesis and signal transduction.
In the coming weeks I’d like to give you an overview of methods in actin research with validated R&D products and kits (actin polymerisation, and G-F actin ratio detection in cells); I also invite you to take a look at a post recently released about actin visualization: Focus on Actin staining and visualization.
In today’s post, let’s concentrate on a method which allows measuring actin binding capabilities of proteins of interest. But it’s not only about the simple fact that a given protein is binding to actin, with the method presented here, you’re also able to get an idea of the functionality of the protein – be it F-actin bundling activity, F-actin severing activity or G-actin binding activity. [Read more…]
Actin serves as one of the major cytoskeleton structures. It is a crucial component 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…]