secretome profile - (Jan/01/2008 )
Hi all, I'm currently looking into a project on profiling a cell secretome. So I'm just looking for general advice on protocols that are available.
1. What kind of methods are there that I can use to profile the secretome? I understand there are 2D gels but are there any other methods?
2. because we have never undertaken such a project before, what kind of things should I be concerned with to set this project up? e.g. sample preparation, concentration of conditioned media, purification etc? I will very likely be dealing with a low volume and low concentration sample (as if it wasn't difficult enough in the first place )
3. What would your general advice be to someone who's just getting into a proteomic type project?
Hopefully someone can find the time to answer this post. Thanks
1. the two major proteomic approaches are 2-D gels based systems and mass-spectrometry based systems. both systems have andvantages and disandvantages. you will need for both skilled mass spectrometry colaborators. my choice would be 2-d gels, although you will see mainly abundant proteins )which are not always the most interesting....
2. what means "low volume and low concentrations" (hoch much is "low" in microgram and microliters?) concentartion should not be a problem, because you will have to precipitate your sample to remove salts and contaminations for 2-d gels. you should use serum depleted medium, otherwise you will see as a major protein serum proteins
3. you will need time. more time than you expect now look for experts in mass-spectometry. establishing 2-d gels is doable. you will need money (for 1.dimension focussing device and IPG-Strips). you should choose a system which is compatible with your SDS-gel systems.
There is an interesting Cell paper "Molecular anatomy of a trafficking organelle." Read this paper, it might help you in your project.
Good luck !!!
Thanks guys for the advice. Luckily there's a proteomics facility across the road that is very experienced in mass spec and 2d gel and they hire out their services. So there shouldn't be too much set up work.