I am conducting a series of assays in which I incubate intact human keratinocytes in culture with a variety of metabolic modulator compounds, then lyse with a gentle buffer and use the lysate for a kinetic plate reader assay for lactate dehydrogenase activity (abcam ab102526). Typically, I immediately proceed to the LDH activity assay after lysing the cells and assessing protein concentration, but for longer incubations I am wondering if there is a safe stop after the lysis step which would be more convenient timing wise. Could I store the lysates at this point and perform the LDH activity assay the next day without losing significant enzymatic activity? If so what storage conditions would be optimal?
Hello Tess McDaniel
You could freeze the lysate at -80 degree C. Do not forget to add a cocktail of protease inhibitors to the cell lysis/extraction buffer before lysing the cells. I would recommend the addition of phosphatase inhibitors as well to the lysis buffer since enzymes are activated by phosphorylation and dephosphorylation may cause a loss of enzyme activity.
Another important point to note is that you should aliquot your sample lysate for future use as multiple freeze-thaw cycles will damage your protein of interest. Storage of protein lysate under such conditions may help for a few months before losing significant activity.
Best.
- Badges
- Science topic
- Similar topics
- Philosophy
- Moral Philosophy
- Free Will