Epitope tags

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Mon Jun 13 11:53:41 EST 2011


In article <27189f3d-4c89-47d8-b9c4-
ee9716bc4e7d from u19g2000vbi.googlegroups.com>, 
novalidaddress from nurfuerspam.de says...
> 
> Dear Engelbert,
> 
> do you have by chance an idea, how your precipitation protocol for IgY
> purification performs in comparison to immobilized 2-mercaptopyrinide
> affinity chromatography (the matrix GE
> - formerly Amersham / Pharmacia - sells for IgY purification?). These
> tiny pre-packed columns (5ml) are nice, but if you start with one egg,
> you already will need for runs, as they only bind 25mg IgY per run.
> Scale up is expensive therefore. I thought of bulk purification in the
> >1g IgY scale (means 10 eggs and up)

I have never used those, hence I can not comment. Precipitation is 
simpleand cheap, but an affinity matrix may give you a much purer 
product. I also suspect that you will have to do at least one 
precipitation before chromatography, as you need to get rid of lipids.

At any rate, I'd treat each egg separately, so you can measure the titer 
of your preparation. Only the preparations with good binding to the 
target protein are used. The simplest way to test several preps in 
parallel is Ouchterlony double diffusion (old-fahioned, but fun). 
> 
> Another question: How to avoid trace contamination with avidin? Is
> extensive washing of the intact yolks in order to get rid of all egg
> white remains sufficient or may I expect some avidin being present in
> the yolk, too?

As far as I know, avidin is in the egg white, which should be separated 
from the yolk before the purification of IgY (ask your mother to show 
you how, it's a standard procedure in the kitchen). In fact, the yolk 
should even be rinsed with PBS before the membrane is punctured.


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