Protease / Phosphatase Inbibitor necessary ? - (Nov/07/2007 )
Dear all
I do expression / purification of potential growth factors in E.coli BL21. I do get protein bands after purification, but cannot check if the function is maintained, because they are new isoforms.
Now I wonder how crucial it is to add Protease / Phosphatase Inhibitor to my lysis buffer?
They did not do it in the lab were I did my diploma, but I would like to know what is common, do you routinely add these for cell lysis ?
I do not know is my proteins are phosphorylated - can someone reccomend a good (and easy !) site for checking ?
I would appreciate your help !
pET
i routinely use PMSF as inhobitor of proteases.
I may add some proteasi inhibitor cocktail for tough proteins but generaly pmsfis sufficent.
In your cas, you may also add phosphatase inhibitor such as NaF. relatively cheap, and may hel discriminate if your ands are different phopshorylation states or not.
I may add some proteasi inhibitor cocktail for tough proteins but generaly pmsfis sufficent.
In your cas, you may also add phosphatase inhibitor such as NaF. relatively cheap, and may hel discriminate if your ands are different phopshorylation states or not.
Thank you for your reply fred 33. What exactly is NaF and could you please explain how I can discriminate if ands are different phopshorylation states or not ?
pET
pET
NaF is sodium fluoride.
As you have proteases and phosphatases in all your cell lysates I warmly recommend always to use inhibitors... as I got crazy over time with always mixing my own inhib. cocktails I started to use complete in combination with PhosStop (both from Roche Applied Science) and I am really happy with them 
As you just have to add the tablet and if you have some leftovers of the stock and can store them frozen it is not only time-sving but also cost-effective...