Concentration of secreted protein in culture media - Concentration of culture media (May/04/2005 )

Hi

Does anyone has a simple solution for problems concerning concentration of large volumes of culture media (baculovirus expression system)? I am looking for a relatively fast method to concentrate culture media (2-3 l) containing my secreted protein. The concentrated solution (100 ml) will be used for purification by Ni-chelating chromatography. Amicon concentrators (Y10 membrane) are sooooo slow...

Thanks in advance

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Hi,

I know this problem, we work with clostridial secretome and compared a lot of different methods for concentration of secreted proteins. Espacially protein-preciptitatin with TCA + DOC can be very effective. But we also get most stable an reproducible results with Amicon ultrafiltration-method.

Tomas

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We used TCA precipitation for SDS-PAGE and Western blots, but this precipitation method gives you denatured protein. We need non-denatured (native) protein for enzymatic tests etc.

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Lyophilisation and ultrafiltration are the general methods of choice for large scale cocnentration. The flow rate in ultrafiltration depends upon the porosity of the membrane. Choice of pore size is decided by the size of the protein. Use the highest possible pore size for ehanced rate.

Ammonium sulphate or acetone ( ice-cold) precipiation can be tried. However, effectiveness of these methods depends upon the protein concentration of the broth.

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I will try ammonium sulphate precipitation. Concentration of my protein in the broth is approx. 10 mg/l. Is this high enough?
Thanks

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To ensure maximal precipitation, it is best to start with a protein concentration of at least 1mg/ml

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Our lab has used Millipore's tangential flow cartridges to concentrate insect cell media. They are relatively quick and allow you to bring the volume of your prep down without precipitating your protein.

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Hi,

May i know if my protein is too dilute, what steps can i take prior to ammonium sulfate ppt? Thanks

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