Ask help for protein purification using His-tag column!

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Wed Oct 23 18:23:48 EST 2002


30 kDa protein pI 5.1 - nothing unusual here. Unlikely to be a DNA binder
due to pI vs size ratio, although I could be wrong. Several things can
happen, for example your protein can have high affinity towards Ni, or may
contain transition metal ions which are being 'sucked out' by incomplete
loading of your resin. pH and salinity can be blamed too - did you check how
your protein behaves in various buffers around pH 6.7-8.5 ?
Another thing to consider is ni-induced aggregation or binding to the column
substrate. It may even be that metal affinity is not suitable for your
protein !

Things to try:

different buffer, salt and pH (within reason). Glycerol, or detergents.
Experiment with a small amount of your protein - how does it react to trace
amounts of transition metals.

Let us know more about the protein maybe we can help you better.

A.G.E.
<chengzhjun at hotmail.com> wrote in message
news:20021022055838.24989.qmail at ww02.hostica.com...
>     my protein is about 30kDa and its pI is 5.1. I used pH=8.0 PBS binding
buffer to banlance the Ni-His tag column. but when i put the protein
solution into the column,the protein solution becomes turbid and the resin
aggregates together and so the protein  buffer could't flow down. How can I
do  with it .
>    Any information would be appreciated!
>   Carol
>
>
>
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