Thursday, September 25, 2014

Using the CLARIOstar and LVF-Monochromator to Identify Wnt Modulators

BMG LABTECH recently posted a new application note (#255) that describes the use of the Leading LightTM Sclerostin-LRP Screening System from EnzoLife Sciences which enables the detection of Wnt-pathway modulators.

This application is important in the study of Wnt effects on bone formation which have been characterized to involve the binding to receptors on bone cells leading to regulation of transcription that promotes bone formation. Specifically, Wnt binds to the LRP 5/6 receptor and the effect of Wnt can be atagonized by Sclerostin when it interacts with the LRP 5/6 receptor leading to inhibition of bone formation. Enzo has exploited the LRP-Sclerostin interaction to produce a system that can identify compounds which block this interaction and could serve as treatments for osteoporosis.

Sclerostin-LRP System Assay Principle


In the Leading LightTM Sclerostin-LRP Interaction System, 96 well plates are coated with Sclerostin and binding of LRP can be seen since LRP5 is linked to the enzyme alkaline phosphatase (AP). After washing steps an AP substrate is added so that LRP5 which remains bound to the Sclerostin on the plates will be revealed by a chemiluminescent signal. Treatments which alter the interaction between Sclerostin and LRP5 can thus be identified based on changes in the luminescent signal which can be detected.


In this application note we found that detection of this chemiluminescent signal can be accomplished in a highly sensitive manner using the LVF-monochromator available on the CLARIOstar®! Using the CLARIOstar® the emission profile for the chemiluminescent signal can be observed. Furthermore, the unique ability of the CLARIOstar® to employ bandpasses up to 100 nm is displayed!


If you would like more information on this and other applications using BMGLABTECH microplate readers please visit the Applications Center on our website!

Wednesday, September 17, 2014

Measuring GPCR signaling with a fast kinetic BRET assay.

G-protein coupled receptors or GPCR's remain a very important subject of study due to their integral role in a variety of physiological processes. For example, the dopamine receptor is a GPCR that is a major target of drugs designed to assist in conditions such as Parkinsons disease. Therefore, continued efforts are being made to monitor the activity of GPCR's in order to identify new treatment options.

In a recent paper in the journal PLOS ONE the authors describe an approach based on bioluminescence resonance energy transfer or BRET to monitor dopamine receptor activation. As with other BRET approaches this assay monitors a protein-protein interaction which in this case is indicative of dopamine receptor activation. A binding peptide was linked to the recently engineered NanoLuc luciferase and a protein that binds to this peptide was linked to the Venus fluorophore. Treatment with agonist leads to association between the peptide and protein which brings NanoLuc into proximity of the Venus leading to a BRET signal.

"Dopamine Electron Map" by Jaelkoury - Rendered on Spartan.
Licensed under Creative Commons Attribution-Share Alike 3.0
via Wikimedia Commons http://commons.wikimedia.org
/wiki/File:Dopamine_Electron_Map.png
#mediaviewer/File:Dopamine_Electron_Map.png
To achieve detection of the BRET signal the authors used a POLARstar Omega plate reader from BMG LABTECH to detect light emitted by Venus and NanoLuc. Furthermore, they were able to perform detection with a 50 millisecond resolution which enabled detailed analysis of activation kinetics in response to dopamine as well as deactivation kinetics.

For more information on the POLARstar Omega and other microplate readers from BMG LABTECH please visit: http://www.bmglabtech.com/

Article citation: J.C. Octeau, et al. "G Protein Beta 5 Is Targeted to D2-Dopamine Receptor-Containing Biochemical Compartments and Blocks Dopamine-Dependent Receptor Internalization" PLoS One20149(8)

Thursday, September 11, 2014

Applications: BRET beta-Arrestin Interaction Assay

Bioluminescence resonance energy transfer, or BRET for short, has been used for many years now by scientists seeking to monitor molecular interactions such as protein-protein interactions.

General bioluminescence reaction of coelenterazine
by Yikrazuul
The original method used Renilla luciferase (Rluc) and yellow flourescent protein (YFP). In the example of protein-protein interactions one protein is labeled with Rluc and another with YFP. When the proteins associate the Rluc and YFP are brought into proximity, such that when the Rluc substrate coelenterazine is present Rluc produces light whose energy can be transfered to the YFP acceptor.

The BRET method has seen several modifications over the years including NanoBRET which was discussed in a BMG LABTECH webinar last year. However, a recent JBC paper employs the classic BRET approach to analyze the interaction of the long-chain fatty acid receptor, FFA4, with beta-arrestin in order to study which ligands bind to FFA4.

This analysis employed the PHERAstar FS from BMG LABTECH. When the FS is equipped with BRET 1 optic module it can perform simultaneous dual emission detection and measure the luminescent emission at 530 and 490 nm produced by YFP and Rluc respectively.

For more information on how BMG LABTECH equipment can help you measure BRET and many other applications please visit our website: http://www.bmglabtech.com/

Original article citation: B.D. Hudson, et al. "The Molecular Basis of Ligand Interaction at Free Fatty Acid Receptor 4 (FFA4/GPR120)" J Biol Chem. 2014 289(29): 20345–20358

Thursday, September 4, 2014

Applications: Screening for Parkinson's disease treatments

Parkinson’s disease is the second most common neurodegenerative disease that affects 7 million people worldwide. It is an incurable, progressive disease with treatments currently restricted to symptom reduction, with debatable efficacy.

TEM image of mammalian lung showing mitochondria
by Louisa Howard
One of the potential causes of the disease is poorly functioning mitochondria in the neurons of affected
individuals. A recent study has also shown that mitochondria are also abnormal in the skin cells of patients. This observation was used to test skin cells derived from Parkinson’s sufferers for potential drugs that can restore mitochondrial function.  Previous compound screens have typically used toxin induced models of Parkinson’s in unaffected cell lines. The authors adopted the approach that even though their model uses skin cells, as they are patient derived they will be more physiologically relevant.

As a proof of principle, patient fibroblasts were treated with 2000 small molecules, and the effect on their mitochondrial membrane potential was measured using a BMG LABTECH FLUOstar Omega plate reader. After performing confirmation experiments, including checking the efficacy on cells from different patients, 15 compounds were found to have a significant effect on  mitochondria. Of these, 2 were taken for further study and were shown to be effective in models of inherited Parkinson’s disease as well as with neuronal models of the disease.


This paper proposes a very interesting method of screening for compounds with efficacy in cells of individuals affected by Parkinson’s in order to relatively cheaply identify compounds worthy of more extensive, neuron focused investigation.

Original article: 
  • H. Mortiboys
  • J. Aasly
  • and O. Bandmann 
  • Ursocholanic acid rescues mitochondrial function in common forms of familial Parkinson’s disease Brain (2013) 136 (10): 3038-3050 

    Tuesday, September 2, 2014

    "Stuck fermentation": an example of prion-based transformation

    Prions are best known for their deleterious effect on higher mammals, for example, in the diseases CJD in humans and BSE in cows. Indeed, BMG LABTECH has provided some instrumenation that has helped with the analysis of these diseases (see application note #232). However, a recent article in Cell, provides an example of a prion-based change that is advantageous, at least to the bacteria and yeast involved.

    S. cerevisiae plasma membrane marked with some membrane
    proteins fused with RFP and GFP markers
    The article entitled: 'Cross-Kingdom Chemical Communication Drives a Heritable, Mutually Beneficial Prion-Based Transformation of Metabolism' describes how bacteria can communicate with yeast to get them to use carbon-sources other than glucose when it is present.

    Typically, there is a very strong preference for glucose use by yeast, when it is present. This is especially true in strains such as Saccharomyces cerevisiae which is why it has been exploited for the production of ethanol. Biologist have known for years about this 'glucose repression' circuit based in the membranes of yeast cells that blocks yeast from using other carbon sources when glucose is present.

    In the current article the researchers describe how diverse bacteria can induce a stable trait, without affecting the DNA of yeast, through the production of protein-based element. This protein fits the characteristics of a prion in that it: 1) is dominant, 2) is inherited in a non-Mendelian fashion, 3) is transferred without the exchange of nuclei 4) does not involve the mitochondrial genome, and 5) employs a molecular chaperone in propagation. This provides an example of a beneficial trait induced by prions that has likely been established to increase the likelyhood of yeast and bacteria survival in a variety of challenging environments.

    Although the use of this prion is beneficial to the yeast and bacteria involved it is a problem for the wine-makers of the world. However, it is believed that now that the means of communication is understood they will be able to combat 'stuck fermentation' by knocking out the bacteria that can trigger the process, avoiding the introduction of bacteria that can induce the process or using yeast strains that are capable of overpowering the bacteria.

    Some information for this blog post was obtained from the Science Daily article: "Prions can trigger 'stuck' wine fermentations, researchers find"

    Journal Reference: D. F. Jarosz, et al. Cross-Kingdom Chemical Communication Drives a Heritable, Mutually Beneficial Prion-Based Transformation of MetabolismCell, 2014; 158 (5): 1083