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How much protein should be loaded to the column during the purificiation?


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#1 Juliasarmoire

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Posted 21 May 2010 - 09:43 AM

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?

#2 Inmost sun

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Posted 25 May 2010 - 01:22 PM

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 :wacko: 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...




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