A growing team of groundbreaking scientists around the world are now sharing their lab notebooks online
The EC50 of Tracer-6908 with ACVR1-c-nanoLuc and ideal conditions for the target engagement assay have been determined in a previous experiment. However, it is still necessary to verify that the IC50 values determined in the assay are closed enough approximation to the actual IC50 values of the compounds. If the IC50 values are strongly influenced Read More …
In nanoBRET assay, a fluorescently labelled tracer binds to the ATP-binding pocket of ACVR1/ALK2. This binding brings the tracer into the proximity (<10nm) of Nano-Luciferase which is fused to the C-terminus of ACVR1/ALK2. Bioluminescence resonance energy transfer (BRET) occurs where the energy released by Nano-Luciferase is transferred to the fluorophore via intermolecular forces. Nano-Luciferase and Read More …
Hello there! This week I won’t be filling you in on my latest experiment, because instead I spent three days at the first ever Cancer Research UK Brain Tumour Conference held in the Royal Society of Medicine in London! (Also the biggest conference I’ve been to so far). The conference gathered over 30 great speakers Read More …
Aside from having high potency towards ACVR1/ALK2, inhibitor compounds ideally should be highly selective and not target other members of the Transforming Growth Factor beta (TGFb) superfamily. TGFBR1/ALK5 is selected for off-target screening because of its potential role in cardiac functions. Firefly luciferase is transcribed downstream of ACVR1/ALK2 in BRE reporter plasmid and downstream of TBFBR1/ALK5 in CAGA reporter plasmid. Renilla luciferase is constitutively transcribed Read More …
Whether or not a cell line can be transfected efficiently is an important factor to consider when designing experiments. Often times scientists would like to deliver into the cells DNA or RNA with the instructions to produce specific proteins or to delete away specific proteins. Transfection via liposome (eg: Lipofectamine 2000) is a relatively simple Read More …
Kinase activity of ALK2 and ALK5 in the cells can be quantified using specific antibodies that bind to target proteins once they are phosphorylated. However, conventional detection in Western blot requires large amount of samples and processing time (proteins are extracted from a large number of cells, linearized, separated in electric field and blotted onto a Read More …
There are concerns that compounds that are effective in inhibiting ALK2 by occupying its ATP-binding pocket might have reduced efficacy against mutant ALK2. That will be undesirable since the compounds should also target the gain-of-function mutant ALK2 in DIPG cells. The following experiment takes advantage of the fact that Activin A activates ALK2-R206H mutant but Read More …
ALK2 and ALK5 are Type I receptors on the cell surface that can be activated by specific ligands (illustrated in the diagram below). Once activated by ligand, ALK2 or ALK5 interact with Type II receptor to be phosphorylated (phospho-group chemically attached to the protein). Subsequently, ALK2 phorphorylates SMAD1/5/8 while ALK5 phorphorylates SMAD2. Phosphorylated SMADs then Read More …
Diffuse Intrinsic Pontine Glioma (DIPG) is a type of brain tumour in the brainstem which is highly aggressive and occurs in children. Treatment options for DIPG are very limited because these tumours do not respond to the chemotherapy drugs currently available for adult gliomas. Whole genome and exome sequencing identified several frequently mutated genes in Read More …
