HIS tag purifiaction
nmckenna at ottawa.com
Mon Jun 12 10:57:08 EST 2000
I regularly purify His x 6 proteins from human cells (I use HeLaT4 and
293T lines). Depending on what kind of proteins you are looking for
(cytoplasmic or membrane-bound) you might want to consider whether
sonication or detergent lysis is best for you. You will need to run
large amounts of cell lysates to get enough protein to see on a western
blot -- I lyse my cells (confluent 100 mm dish, transfected) in 1 mL of
detergent buffer (i work with trans-membrane proteins) and have to run
50 uL of it to see on a western in crude lysates. My elution volume is
usually 100-250 uL. (I transfect transiently, though, and my proteins
are not secreted!). I often follow the qiaexpressionist handbook
protocols, but Qiagen's product literature (I think it's called Qiagen
Notes) sometimes has notes on new protocols.
I hope this helps...Good luck!
Marc Busch wrote:
> I'm trying to expressed my protein fussed to a his tag on its C term.
> This will be expressed in mammalian cells (HeLa, 293T etc). Can any
> recommend a protocol / kit for the purification? Thanks in advance
> Marc Busch
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