I am working on a secreted protein, so my protein samples are taken from the cultured media. I wanted to check interaction of this protein with other proteins also, so I am planning to do co-immuno precipitation. However, so far I haven't found any media protein extraction protocol that can be used specifically for co-IP (if any?). For western blot, I just use TCA-NaDOC method to extract protein from media but I am thinking maybe some factors in the extracting step might affect the co-IP process so any suggestions would be helpful.
Hi Richelle,
TCA is quite rough and might lead to permanent changes in proteins. I prefer ammonium sulphate ppt.
If the reason for the precipitation is only to concentrate the media, you might also consider other ways to concentrate, It maybe can be done with centrifugal tubes like Amicon or vivaspin.
Another option is to omit the centrifugation and just add a larger volume of culture media to the co-immunoprecipitation beads. This combined with a longer incubation time.
Good luck
Ultrafiltration is one way to concentrate samples. We run large volumes of media over a hydrophobic column which can be eluted with distilled water. This can be done at any scale by varying the column size. In some cases you may need to add ammonium sulfate to the medium to get the protein to stick to the column.
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