Histidine in buffer, what is the role? - cytosolic and membrane protein isolation (Nov/15/2006 )
1. I have read several protocols for isolation of membrane protein/enzyme. There is one that I like to use it for my work but there is still one thing I did not understand why the author use histidine in his homogenization and some other buffers. Here are the componets of the buffer he used:
10% sucrose, 20 - 30 mM histidine, p 7, 0.5 mM DTA, 0.23 mMPMSF, 0.83 mM benzamidine.
Can anyone explain me the role of histidine in that buffer please!?
2. I want to modify the buffer a little bit to fit my own purpose and here is my proposed bufer:
HEPES Hepes-tris: 10 mM
EDTA: 1 mM
KCl: 25 mM
Sucrose: 250 mM
DTT: 0.5 mM
PMSF: 0.23 mM
Benzamidine: 0.83 mM
Can you please also tell me that if the presence of these protease inhibitors would interfere with my protein and Ca2+-ATPase assays?
Thanks a lot for your time and help!
histidine is the buffer component, better try a histidine-imidazole buffer than histidine-tris-hepes buffer