For protein extraction I am using the Roche cOmplete Tablets , Mini EASYpack. The instructions suggest dissolving one table in 10 ml in H2O or phosphate buffer. However, it does not mention how much I should then add to my lysate. Has anyone used these tablets as protease inhibitors for protein extraction? If so, is the volume required, dependent or cell number of volume of lysis buffer?
I usually dissolve the protease inhibitor cocktail in the lysis buffer, then use it to lyse the cells.
Hello, Randeep,
1 tablet is good for 10 ml cell lysate with normal proteolytic activity. Youc can directly add the tablet to your extraction solution (cell lysate). You may use more 1 tablet depending on the proteolytic activity of the extraction solution.
Hope this helps.
Best,
Arizaldo
Generally I used the solutions from sigma and I always added it to the extraction buffer 1/250.
I dissolve 1 tablet (thermo fisher) in 2ml DI water and use 0.4ml of this in 6ml of lysis buffer that is good for 100ml culture pellet E.coli. You can store the remaining at -20 degree.
I always prepare a higher conc. Stock which is more stable (if stored at -20°C). From that stock (7x conc) I will take the volume necessary to have a final 1x conc. in my lysis buffer. Alternatively, you van just add the tablet to your lysis buffer directly.
Cheers,
J
To the PIC cocktail you further can add 25 mg leupeptin, same DMSO dilution