How much protein should be loaded to the column during the purificiation? - Is less more? ;) (May/21/2010 )

Just a quick question... I'm using Protino Ni-TED 1000 columns to purified a histidine tagged protein.. the maximum binding capacity is 2.5 mg. Now I have to ask how much protein I should load to the column to get that 2.5 mg of protein or even close to it Is it better to overload the column for example with 10 mg of protein or for example to load only 2 mg of the protein?

Also which one is better to have high concentrated protein sample or then less concentrated meaning should the liquid passing through the column be a lot or less... or does that even matter?

-Juliasarmoire-

Juliasarmoire on May 21 2010, 05:43 PM said:

Is it better to overload the column for example with 10 mg of protein or for example to load only 2 mg of the protein?

Also which one is better to have high concentrated protein sample or then less concentrated meaning should the liquid passing through the column be a lot or less... or does that even matter?

as you do not seem to have a running protocol, you have to optimize the conditions; binding capacity of column matrix means in most cases "specific binding capacity"; is your protein of interest one fourth of the crude mixture, you may start with 10 mg; overloading will only increase impurities...

-Inmost sun-