Isolation of secreted proteins from culture media
Posted 18 June 2009 - 07:59 AM
I'm a first year PhD student and I'm trying to come up with a protocol that I can use to isolate secreted proteins from the media of a 2 day culture of oomycete mycelia. I know that I need to filter the media through a membrane to remove the hyphae from the media before I can precipitate the proteins but I was wondering if anyone is aware of anything that I can do to reduce the background from the media itself prior to the precipitation step?
Posted 18 June 2009 - 09:42 AM
What do you mean by "background"?
If you want to reduce volume and remove some of low-MW components you may try ultrafiltration (could be quite costly) or dialysis-concentration combo (ie. first dialyse then place bag on ficoll, high-MW glycol or dry sephadex). To reduce volume only you may simply freeze-dry (lyophilize) or even vacuum rotary evaporator.
To remove other proteins it would require to know their properties eg. molecular weight, pI or affinity and I think it would be easier to actually catch your protein than remove other. (This may be an idea for you to explore - eg. if culture medium does not contain too much salt, you may try to catch your protein on ion exchange resin/medium.)
Posted 18 June 2009 - 10:49 AM
Yes, this is important that you should know the composition of the media that you are using because in the end you may be getting many proteins that are actually from the media and not the ones that are secreted. I have tried doing this many times with mammalian cells and indeed its very difficult since I was mostly getting BSA band (from the serum) on my gels and blot. I would suggest that you shift to a special media (available from certain companies) that contains no or relatively less proteins.