Thursday, June 26, 2014

Application: Monitoring hydrogen peroxide production in cell monolayers

Hydrogen peroxide (H2O2) has been characterized for its role in a wide variety of biological process. Some beneficial, such as regulation of immune system function, and others viewed detrimental, such as involvement in aging. Therefore, there are many instances where understanding the cellular level of H2O2 would be useful. Application note 252, recently added to BMG LABTECH's website describes the use of the PHERAstar FS to detect a genetically encoded redox probe specifically designed for H2O2 detection.

Lung adenocarcinoma H1975 cells expressing the cytosolic
roGFP2-Orp1 probe. Cells were exposed to hydrogen 
peroxide as indicated, and the probe response followed using 
the PHERAstar FS
This note entitled: 'Real-time monitoring of genetically encoded redox probes in mammalian cell monolayers' describes a very useful tool that can be used to monitor cellular redox changes. This note employs biosensors based on redox sensitive green fluorescent protein (roGFP). RoGFPs are GFPs that have had 2 surface exposed residues changed to cysteines. These cysteines are in appropriate positions to form a disulfide bond. In the oxidized state this bond is strained and the protein contains localized structural changes which are believed to be responsible for the spectral changes of roGFP that change the excitation maxima from 485 nm to 400 nm. Furthermore, roGFP can be engineered to respond to specific redox species. In this case,  fusion to the thiol peroxidase Orp1 generates a H2O2 sensitive roGFP.

The PHERAstar FS proved to be an excellent tool for detecting changes in H2O2 using this biosensor. To find out more please visit the Applications Page on BMG LABTECH's website.

Application note: #252 Real-time monitoring of genetically encoded redox probes in mammalian cell monolayers