MICROPLATE READERS
FIG. 1. Schematic of PROTAC function. PROTACs simultaneously bind an E3 ligase and a target protein to form a ternary complex. The target is then poly-ubiquitinated and degraded
a dose dependent relation between PROTAC concentration and their ability to bring BRD4 and E3 ubiquitin ligase together (Fig. 2b).
CONCLUSION Te PROTAC-induced combination of E3 ligases and proteins to prepare their degradation is a cell-based process with many variables. Terefore, it is best
monitored directly in cells and as it happens – in real-time. To this end, a stable cellular experimental setup as well as reliable detection is needed. Te presented BRET assay is a ratiometric method and stable to fluctuations in cell number or luciferase expression. Tis is achieved by relating the signal of interaction (fluorescence) to an internal control (luminescence indicative for luciferase expression and cell number). Te detection device requires not only a robust and sensitive measurement of signals, but also a homogenous temperature of 37°C inside the measurement chamber and a 5% CO2
FIG. 2. (a). Principle of BRET-based live cell assay to monitor PROTAC activity. The target protein is marked with a luciferase while E3 ubiquitin ligase is marked with a fluorophore. If both are brought together, energy transfer takes place and BRET ratio increases. (b) BRET assay employed to monitor the interaction of BRD4 and VHL induced by the PROTAC ARV 771. Indicated PROTAC concentrations were added to cell system and signals were recorded on a Clariostar Plus microplate reader
atmosphere to stabilise the pH in bicarbonate-buffered cell media. Te Clariostar Plus microplate reader combines stable measurements with atmospheric control and proved to be well suited to measure the real-time PROTAC assay (Fig. 2).
Dr Ann-Cathrin Volz is applications specialist at BMG Labtech.
www.bmglabtech.com
OPTIMISED MICROPLATES FOR NUCLEIC ACID PURIFICATION P
orvair Sciences’ new medium and high bind Krystal microplates have been designed to set a new standard for uniform and reproducible capture of a wide range of biomolecules ready for affinity binding assays such as ELISAs. Manufactured from high-quality crystal
polystyrene, these new automation-ready plates are available in a choice of ANSI/ SLAS standard (96-well and breakable 8-well strip) formats. Offering working volumes up to 320 µl the new Krystal binding plates incorporate a flat bottom to ensure superb assay detection every time. Designed for hydrophobic passive
adsorption of lipid-rich large biomolecules and antigens, including viral antigens, medium binding plates offer a binding capacity of approximately 100 to 200 ng IgG/cm². The high binding plates are optimised
for passive adsorption of proteins with different grades of hydrophilicity. Offering
a binding capacity of 400 to 500 ng IgG/ cm² these high bind plates are ideal for immunoassay of glycoproteins and serum samples. Krzysztof
Kielmann at Porvair Sciences comments that, “The aim of binding assays is to measure interactions between two molecules, such as a protein binding another protein, a small molecule, or a nucleic acid. We have developed our latest range of Krystal binding plates for laboratories seeking to uniformly and
reproducibly capture biomolecules, thereby enabling them to improve their assay detection results.”
For more information visit
www.porvairsciences.com
www.scientistlive.com 23
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60
