Protein purification - (Jul/23/2008 )

Dear all,
Please help me with the protocol for protein/enzyme purification
Thanks

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If you have His-tagged proteins you can follow this protocol from Qiagen. They have information abotu purification under native and denatured conditions. In my experience, if you are purifying an enzyme, the purification should be done at 4 degrees C.

http://kirschner.med.harvard.edu/files/pro...ssionist_EN.pdf

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Hi,
Thank you very much

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What about isolating secreted proteins, that is very small 38kDa? I try to isolate a protein that is secreted by a bacteria. We don't have antibodies for this protein so Western blots is out of the picture. Do you have any suggestions?

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I presume your protein doesn't have any tags on it.
You can concentrate the media by transverse flow, or you could do step-wise precipitation with ammonium sulphate and look at the fractions for activity. Either way, you need to reduce the complexity of the sample before you have a chance of seeing where your protein is.

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