There are very few assays to IDENTIFY proteins unless you have specific proteins of interest and have antibodies for them. Gels can give you approximate molecular weights, but bands and/or even spots typically cover at least several proteins, and the molecular weights are approximate (e.g., a gel spot or band might correspond to a molecular weight of, say, 15,000, while mass spec would give more like, 15,201.2). Mass spectrometry is often the start, and then Westerns, for example, would be used to confirm identifications of interest.

(But nothing in this area is 'simple'!)

:)

Salt precipitate supernatant and do commassie blue to identify by size

Hi Pastula

I think the FACS analysis is the best way to go if you are looking for proteins in exosomes using beads or western blotting with specific antibodies. ELISA is the other way of identifying specific proteins

RKG

There are very few assays to IDENTIFY proteins unless you have specific proteins of interest and have antibodies for them. Gels can give you approximate molecular weights, but bands and/or even spots typically cover at least several proteins, and the molecular weights are approximate (e.g., a gel spot or band might correspond to a molecular weight of, say, 15,000, while mass spec would give more like, 15,201.2). Mass spectrometry is often the start, and then Westerns, for example, would be used to confirm identifications of interest.

(But nothing in this area is 'simple'!)

:)

I agree with John. You can try few simple techniques such as FACS analysis, at the same time you can also go for ELISA and western blot. I believe western blot would be more simplified if you have the antibody for it. :)

Hi, thank you all for the answers!

The supernatant causes an effect on cells I am working with and we would like to know which protein is resposible for that and then go for the signalling pathway.

At the moment I do have only hypothetical protein candidates,

so I do not have my protein of interest yet.

The mas spec is planned. I was wondering whether I could treat the supernatant in different ways to speculate for the general features of the potein of interest e.g. if contains lipid modifications etc?

go for dot blot analysis..

If you have no ideas of what protein you are looking for, there are different targets/pathways related protein arrays which are commerical available (Eg. angiogenesis array, phospho-protein array supplied from R&D, Raybiotech, etc).

If it is a quantification of total protein, then Bradford protein assay will work.

I think at least you can concertrate the supernatant and run a SDS-PAGE, so that you can estimate the weight of proteins and filter your candidates.

Please be careful with the MS results, since if your protein will be at low level the abundant contaminants will cover it. Anyway the MS is a very, very semi-quantitative at this moment, thus your protein of interest can be not found as regulated or you will find an artifacts. If you have already candidates, please buy the antibodies, and verify it by ELISA or Western-blot. If these analyses will show no regulation you still can publish it as an negative control…

I think that given you have a bio active response the first step would be to fractionated your supernatent using simple size exclusion chromatography using something like sephacryl S200. Test each fraction against the target cells for a biological response. This will give you a rough idea of the globular protein equivalent molecular mass of the protein (not all secreted proteins are globular and irregular elongated proteins elute early because there effective dynamic radius is that of a much heavier globular protein). You can refine the isolation by using s100 or s300 if it is a particularly small or large dynamic radius protein. More importantly the fractions with bio activity can then be subjected to mass spectrometry in the hunt for the target.

However, I would suggest growing the producer cells in serum free media for a 2-3 days and using this in your fractionation and mass spectrometry experiments. Be aware that bovine serum albumin is so abundant it can swamp out signals from less abundant target secreted proteins - hence trying serum free media may really help in your identification of candidate proteins.

Dear Agnieszka

The best way to find a protein that is responsible for an action in cell signaling is proteomics study.

You can run a 2DE to find over hundred protein spots and then analyses them one by one.

For total protein measurement, you can also use Bradford protein assay in simple three steps.

Cheers

I would like to thank all of you for very helpful answers!