[Protein-analysis] Re: Batch vs column chromatography

Julio juliocortazar at o2.pl
Fri Mar 31 01:35:56 EST 2006


Dear DK,



Which means your expression level is very low.

Well i suppose it might be becouse we use pTrc plasmid and E.coli
DH5alpha strains, they are not new... Recently we had an idea that our
protein is not properly exported to the periplasm - it has signal
sequence from Salmonella strain which and this sequence is probably not
cleaved when protein is expressed.

But why 12 hr?

We load the protein so long 'cause when we tried to load it in about 3h
our yield  was lower and more protein was passing the column not
binding it.

Going back to your orignal question, re: batch. No, at this
expression level, if you go with 150 ml lysate per 1 ml sorbent,
you'll liely end up losing >50% of your total expressed protein
in the unbound sup.

So what do u suggest is that column purification is better in this
circumstances of low level of expression that batch method.

JC
> DK



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