I have ordered the SOD colorimetric kit (invitrogen) and a GSH/GSSG ratio assay (abcam, ab205811) and was hoping to use the same brain tissue for both kits. In the methods provided the by the manufactures, the GSH/GSSG assay requires PBS/0.5% NP-40 and the SOD assay just requires PBS. Does anyone have experience with these kits and can advise? Could NP-40 be left out if cells are throughly homogenised with a ultrasonic homogeniser since this is just a detergent?
Really appreciate any advice.
Hello. I have not used these kits, but in my opinion. You can leave the detergent since you are using sonication and of course I suppose you know which SOD you want to extract. Meantime I advice you to try using the two types of buffer i.e. in the presence and absence of the detergent, then compare the activities that you obtain in the two cases then decide which one give better result. Of course you can at the same time try to run electrophoresis and stain for the activities to look for the variation in the forms of the enzymes you obtained in both cases, by this you will confirm what you will obtain.
Good luck.
Be careful. Although you may achieve sufficient cell lysis to release cell's glutathione and enzymes into the medium, glutathione oxidizes very rapidly in neutral pH. I am not sure about the specific kit you mentioned, but a common practice in measuring glutathione with colorimetric methods is to prepare the cell lysate in acid (e.g., trichloroacetic acid or sulfosalicylic acid). The use of acid serves two purposes, 1. to keep the pH and avoid oxidation, and 2. to precipitate proteins that might interfere in the assays.
See: https://onlinelibrary.wiley.com/doi/10.1016/0307-4412%2883%2990048-1 and Article Assay for quantitative determination of glutathione and glut...
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