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Trouble concentrating protein for cell culture media


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#1 monosyllabikgurl

monosyllabikgurl

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Posted 24 July 2011 - 11:04 AM

Hi,

I have been trying to concentrate protein from conditioned media from primary fibroblasts with some difficulty. After collecting the media, I did a protein assay (BioRad DC) and, not surprisingly, the protein concentration was very low (approx 0.1 mg/ml but some undetectable). I have tried using the Amicon Ultra-15 MWCO 10 kDa to concentrate approx 5 mls of media. I spun the media at 4,000xg for 60min and obtained approx 200-250 ul of media however after reading the protein concentration again it was only a max of 0.2 ug/ml. . .

I dont think that I will have lost protein due to over-concentration as it was so low in the first place, but I am assuming that some has stuck to the membrane in the concentrator. I have read about pre-washing first but does anyone have any experience of this improving the yield and possibly a protocol? Should I spin for less time? I aim to do Western blotting for particular proteins with the media. I have already performed these westerns on tissue extracts and I needed to load 50ug of protein. Obviously I wont be able to get the protein concentration this high in the media but clearly I have a long way to go.

Any help would be appreciated. I have also tried TCA ppt however this was worse than the centrifugal concentrators, and also not ideal as some of the proteins I would like to assay for later need to not be denatured.

Any help would be appreciated.




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