anti-His6 as a mammalian probe ?

D.K. dk at no.email.thankstospam.net
Wed Nov 28 21:21:48 EST 2001


"Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote:
>Thanks, Dima (I cracked your disguised identity after your mentioning
>Osterman's book earlier :-))

Heh, Emir - hard to make a mistake :-) If it were up to me, I'd make 
Osterman a required reading to every student approaching a chromatography 
column closer than couple meters. 

(Not really a disguise but I am getting way, way too much spam than I 
could afford or my daughter accidentally see if I forget to delete it). 

>A relevant question. Does it really matter where to put 6his: on C-term.,
>N-term, or internally? 

I know Sigma stuff binds equally well to N- and C-term, and there is absolutely
no reason to believe it won't bind _exposed_ internal sequence. If you 
are absolutely sure about the loop to put it in, all should be well. 
(But in any case inserting something in the middle runs higher risk of 
screwing everything up). 

>I cannot put it on termini because N-terminus is a
>binding sequence,

It still _might_ tolerate something in front maybe? 

> C-terminus is a protein transduction domain (to allow
>peptide to penetrate live cells and tissues) that should be on the terminus.
>So, the only place for a tag is in between. I noticed that Qiagen has 3
>different Ab's for C-, N-terminal and for internal 6his. What's the story
>here?

I'd have to look up their brochure but I feel you misread. I remember 
their "penta" advertised as binding everything. 

>Another question, would introduction of say 8His increase efficiency of
>antibody recognition (it seems it might improve affinity purification as an
>added bonus)?

Don't know. If the tag behaves, 6His already gives very high affinity.
If it does not, having more sites to bind might help, but I suspect 
in most such cases the problem is elsewhere (buried inside, interacts
with protein itself, ect). 




More information about the Methods mailing list