I am planning on purifying a small 12 kDA protein with a 6x-His tag using Ni-NTA resin.
Many protocols recommand the addition of protease inhibitors like PMSF for optimal lysis and purification of the protein.
However, I will be needing to remove the his-tag using thrombin once the fusion protein is purified. Will PMSF prevent me from doing so ? If so, what are my options ?
PMSF is unstable in water. By the time you are ready to do the thrombin cleavage, any PMSF added during lysis and purification should have broken down.
Yes PMSF will inhibit thrombin. Depending on the specifics of your purification process, you may skip the addition of PMSF during the last purification step or simply do buffer exchange against PMSF-free buffer using e.g. a PD-10 desalting column.
Hope it helps!
Dear Martin
generally we add the protease inhibitors just in the lysis/ binding buffer and not in the washing and elution buffers for imac because once your protein is eluted, it is quite pure and if it's folding is stable generally we do not observe degradation.
however it also depends if thrombin
work in presence of high imidazole concentration because if not, you have to change the buffer and as Alejandro suggest you can do it by sample concentration with amico ultra and pd-10 desalting.
Sincerelly I'm a fun of Tec protease and I'm not an expert of thrombin.
in all cases after thrombin digestion you can exchange again the buffer by pd-10 and perform a 2nd subctractive imac step to remove the cutted tag and the thrombin if it has an his tag.
if you are interested to know more details about subctractive imac and desalting with pd-10 you can find a video on my blog: ProteoCool ( https://proteocool.blogspot.com/)
ProteoCool n°11(Rapid buffer Exchange by PD-10
ProteoCool n°13(Subctractive IMAC)
available at page3
good luck
Mamuele
PMSF is unstable in water. By the time you are ready to do the thrombin cleavage, any PMSF added during lysis and purification should have broken down.
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