Hello,
I am expressing wt. BRD4-D1 (only first bromodomain of BRD4 protein) using BL21 cells. During cell lysis with lysis buffer and lysozyme, I add a saturated solution of PMSF in 1 mL of methanol and no protease inhibitor cocktails. Is PMSF alone sufficient? I am not getting the full length protein. I think I am getting truncated proteins during my purification using IMAC.
PMSF is a protease inhibitor. You may need a few more protease inhibitors or purchase protease inhibitors cocktail from Sigma Aldrich.
PMSF is a serine protease inhibitor, which works well also by itself. It is poisonous so people tend not to use it anymore. Obviously is not the standard for all protein expression and occasionally you will find proteins that get degraded, in that case it is worth to test a inhibitor cocktail as the Complete EDTA Free (if you use Ni-Column). Alternatively you can use a mixture of PMSF and benzamidine. Good Luck
Hello,
Thanks for your suggestions. I use a saturated solution of PMSF. I also keep adding more PMSF to my cells..Like if I do two sonication cycles, I add more PMSF after one cycle is complete.
You should use no more than 2mM PMSF to inhibit serine pro tease and you can add dithiothreitol (1 mM) to inhibit thio-protease as final concentration and be sure they are soluble in your the buffer you are using and since PMSF is insoluble in water so when you prepare it you dissolve the weighted PMSF in few drops of methanol, or ethanol then complete the volume with distilled water to obtain the required concentration . So you can not use the saturated solution of this inhibitor as you did.
Hello Hathama, can you explain why do you say that we shouldn't use more than 2mM? Is there any side-effect associated?
This is the recommended concentration ,if you have a look at the protease and their different classes you will find out the suitable concentration which you should use.If you have ask if there is any side effect ,i believe that when they suggested this concentration this is based on several exp. that they had done and they found out this is the best one& I believe this is the reason that we do the literature survey to get benefit from others work & recommendations.
- Badges
- Science method