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Histidine in buffer, what is the role? - cytosolic and membrane protein isolation (Nov/15/2006 )

Dear All,

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

-The Bearer-