metall ion affinity purification of proteins - something better
Dr. Duncan Clark
drc at nospam.demon.co.uk
Thu Jul 6 05:06:09 EST 2000
In article <200007022145.XAA09296 at intwww.zit.med.uni-tuebingen.de>,
Wolfgang Schechinger <Wolfgang.Schechinger at med.uni-tuebingen.de> writes
>are there other (competitive/better) methods for purifying proteins
>using affinity to chelated metal ions than his6 / nickel?
Yesterday I had a student run a comparison between Co charged IDA and Ni
charged IDA using the same crude extract of an N-terminal HIS tagged
yeast PPiase expressed in E.coli.
The Cobalt column gave a higher final yield, approx double by protein
gel. The columns were not overloaded in terms of HIS tagged protein but
the Co column appears to have better affinity for this protein than the
Ni. The difference in yield was accounted for by unbound protein in the
flow through of the Ni column. Both gave the same rough final purity in
terms of other proteins showing up in the elution buffer, at least 95%
pure by eye.
It's only a one off experiment but in future we will now compare Ni and
Co before we scale up to 200ml columns etc.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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