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recombinant protein purification

recombinant protein purification - Biochemistry Forum

recombinant protein purification - Discuss and post questions regarding the study of Biochemistry. If you need homework help this is the place to ask!


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Old 01-29-2011, 03:41 PM
Pipette Filler
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Question recombinant protein purification



Hi
A question about affinity chromatography (recombinant His-tagged protein purification with an Akta system). Please suppose I use an 1 mL column (nickel), I load my clarified extract, I wash and I perform the elution with a linear imidazole gradient. I get a peak in B5 fraction (for instance..).
Now, I want to scale up : I perform the purification exactly in the same conditions but I use 5 times more extract (5L culture instead 1L before) and I set on the system a 5 mL column (exactly the same type).
The question : in which fraction will I find my peak ? B5 ?
Thank for your help in advance:
Best regards!
Nico
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Old 01-31-2011, 06:56 AM
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Default Re: recombinant protein purification

Hola, Yes if you mantain the same program the same wash volumes and the same fraction volumes. Only if the colum is saturated you will see a part of your protein in the flowthrough, but knowing the amount of purified protein in small scale you could know with that volume it will be saturated. Depending of the protein the capacity of column is 20-40mg. Buena suerte
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