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.

Hi to you all.

I do agree you will not have problems with PMSF which was added during lysis/binding buffer step. If you find out you have problems of degradation after your first purification round, you may add it once again in your elution buffer or after it.

There is lots of nice protocols where, depending on a few factors, you may perform thrombin cleavage on collumn. Check it!

http://wolfson.huji.ac.il/purification/TagProteinPurif/CleavTombin.html

Hope it helps.