Kinase experiment:

Monochlorobimane (mCB) is added to the second half liver tissue homogenate to a final concentration of 100 mM along with glutathione S-transferase (1 U/mL) obtained from equine liver; the homogenate is then allowed to incubate at room temperature for 30 min. The Glutathione (GSH)-Monochlorobimane adduct is measured in a microtiter reader[1].

Monochlorobimane is a fluorescent dye (λex=380 nm, λem=470 nm).

A common technique to measure Glutathione (GSH) in cultured cells is to add Monochlorobimane to the culture medium where it readily enters cells to form a fluorescent GSH-monochlorobimane adduct that can be measured fluorometrically. It is clear that the Monochlorobimane approach gives comparable results to the HPLC technique. Although most laboratories use 100 mM Monochlorobimane in their assay our results show that the Monochlorobimane concentration can be lowered to 25 mM and still accurately measure homogenate GSH content[1].

[1]. Kamencic H, et al. Monochlorobimane fluorometric method to measure tissue glutathione. Anal Biochem. 2000 Nov 1;286(1):35-7.