DNA and Histone proteins are targets for epigenetic modifications.
One of the roles of Epigenetics is to drive cellular differentiation from totipotent Stem cells to fully differentiated cell types, by regulating gene expression through patterns of modifications that differ according to the cell type and the cell status. This means that the patterns may significantly differ between stem cells, germ cells, and differentiated cells.
It has been found that cancer cells often differ in regard to specific Epigenetic modification sites too. Thus, epigenetic enzymes are considered as potential pharmaceutical drug targets or biomarkers, and quite some effort is made to define drugable targets.
In a previous post I focused on histone modifying enzmes (writers) . Today I will concentrate on those enzymes which erase modification from histones (erasers). Later on, in a further post we’ll be focusing on proteins which recognize specific modifications (readers).
Human plasma samples assayed for ADAMTS13 activity. Human plasma was spiked with ADAMST13 inhibitor (1,10-Phenanthroline). Inactivated human plasma was spiked with recombinant ADAMTS13. Samples were incubated at 37°C for 1 hour and fluorescence was measured at Ex/Em=490/520nm (FlexStation 384II, Molecular Devices).
ADAMTS13 (A Disintegrin-like and Metalloprotease with Thrombospondin type-1 motifs 13) is a metalloproteinase that is primarily synthesized in liver cells and secreted into blood circulation. ADAMTS13 specifically cleaves von Willebrand factor (vWF) at the Tyr1605-Met1606 peptide bond. vWF is a multimeric glycoprotein that mediates adhesion and aggregation of platelets. ADAMTS13 is essential for preventing platelet aggregation in the circulation. Deficiency of plasma ADAMTS13 activity may lead to thrombotic thrombocytopenic purpur (TTP), a potentially lethal syndrome characterized by the formation of VWF-platelet-rich thrombi in the arterioles and capillaries. Studies have shown that ADAMTs13 is also involved in angiogenesis, inflammation, atherosclerosis, and liver cirrhosis. Measurement of ADAMTS13 activity in plasma has been used for supporting diagnosis of acute TTP.
New kit to measure the activity of ADAMTS13.
Developed by AnaSpec, this new kit employs an internally quenched vWF73 FRET peptide substrate for the detection of enzyme activity. This substrate contains a novel 5-FAM/QXL® 520 FRET pair. ADAMTS13 cleaves this FRET substrate into two separate fragments resulting in the release of 5-FAM fluorescence, which can be monitored at Ex/Em= 490/520 nm. The long wavelength fluorescence of 5-FAM is less interfered by the autofluorescence of components in biological samples.
The homogenous assay (mix-and-read format) can be completed in as short as 1 hour.
Interested? Leave your comments below, or let us know if you’d like to learn more about the kit and its use.
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.
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.
Measuring the activation of small G proteins
In the past, the activation of small G proteins (i.e. the transformation of the GDP-bound form to the GTP-bound form) could only be measured in pull-down assays. The assays make use of effector proteins which specifically bind to the activated forms of small G proteins. Coupled to beads these proteins can be used to pull-down the activated small G protein from a cell lysate.
Cytoskeleton Inc. introduced a new format of this approach a few years ago. With the G-LISA technology, the effector proteins which selectively bind to activated small G proteins are coupled to 96 well plates, making it possible to run an ELISA-like activation assay. To help you to choose the right activation assay format for your application, take a look at this short video.
New trial sizes for the most commonly investigated small G proteins
Up to now, researchers had to order full 96 well plates to start using the G-LISA technology.
Now Cytoskeleton Inc. have launched trial kits (which are attractively priced) to enable you to establish the method in your lab:
Measuring soluble collagen in biological samples is a straightforward experiment which can be done with the Sircol soluble collagen assay using Sirius Red dye (Sircol dye) in a convenient manner (we explored this method in a recent post). Nevertheless, up to now it has been a challenge to measure insoluble collagen.
