Dear Madisson, you can read this protocol:

http://www.ncbi.nlm.nih.gov/pubmed/25458555

However, I would suggest you to use direct in situ immunolocalization. Fraction isolation always raise a questions how pure is your fraction. There are a lot of protocols for in situ membrane localization. Best wishes!

If you wish to do fractionation the protocol in this paper works well

Germain, H., Gray-Mitsumune, M., Houde, J., Benhamman, R., Sawasaki, T., Endo, Y., Matton, D.P. 2013. The Solanum chacoense ovary receptor kinase 11 (ScORK11) undergoes tissue-dependent transcriptional, translational and post-translational regulation. Plant Physiology and Biochemistry. 70, 261-268.

Dear Madisson,

If you cannot rely on immunolocalization because you don't have an antibody that works well for this matter, fractionation is indeed a good and cheap method. In any case, however, you need to have an idea of the density of the vesicle you are interested in. Hence you can adapt the sucrose density to that of the vesicles you want to recover from the following procedure: http://www.abcam.com/protocols/subcellular-fractionation-protocol.

Anyhow, you need to keep in mind that this approach is only a way to get an enriched fraction in the desire population of vesicle and needs to be performed very carefully to avoid vesicle damage.

Dear Madison,

as Taras mentioned fractionation will not be very informative on identifying your compartment as it is quite difficutl to obtain pure fraction of endomembrane compartment.

If you want to prove your protein is indeed membrane associated i would suggest a simple microsome purification (separating soluble protein from membrane proteins by an ultracentrifugation at 100 000g and analyzing the pellet versus supernantant by PAGE-Western blot) this will help showing your protein is indeed associted with membranes.

As to identify its exact compartment, it would be easier (and show a clearer result) to colocalize it with known published endomembranes markers by either transient or stable expression (depending on the system you work with).

good luck

Dear Maddison,

Subcellular fractionation might be cheaper, but in my experience is not an easy experiment to set up and is quite difficult to specifically isolate specific types of vesicles. There are several protocols available for exosomes (with and without the use of sucrose gradient), while if you're interested in endosome separation, you can have a look at Alanko et al., Nat Cell Biol 2015, where they used subcellular fractionation to separate plasma membrane, cytosol and endosomes. Here is an extract from their methods: "Briefly, adherent cells were scraped on ice into hypotonic lysis buffer (10 mM HEPES-KOH pH 7.2, 0.25 M sucrose, 1 mM EDTA, 1 mM MgOAc and protease and phosphatase inhibitors (PhosSTOP and Complete mini tablets from Roche)). Cell membranes were fragmented with a French press and nuclei removed with 10 min 1,000g centrifugation. The plasma membrane fraction was collected with 10 min 10,000g centrifugation and endosomal/cytoplasmic fractions with 1 h 100,000g centrifugation. Membrane fractions were washed at least once with lysis buffer and cytoplasmic fractions were centrifuged twice. All fractionation steps were performed at +4 °C or on ice. All fractions were dissolved in sample buffer for immunoblotting."

Hope this helps,

Best

Elena

Thank you everyone for your help!

First I ran the experiment on my cells to establish a fractionation assay that actually works and then I will be doing it on my microparticles with hopes it works with those as well. however from the sounds of it I may need to change the sucrose concentration depending on my microparticle density.

I tried running it using a protocol that worked off the abcam protocol except it didn't pass the samples through a 25 gauge needle. I got an fairly large pellet for the nucleus spin but no pellet for the membrane spin (ultracentrifuge 100,000 x g) spin. Could I have lost the membrane proteins in the the first spin?

Kian, the protein I am looking for is a proteoglycan that is in the extracellular matrix and has been shown to be anchored to heparin sulfate proteoglycans like perlacan, versican and agrin. Those three are all extracellular matrix proteins that are bound to the membrane on cells. Could I do a co-ip with my microparticles and show that if the TSP-1 is bound to those proteoglycans and conclude it that way?

Lastly, Elena, in your protocol you sent it mentioned that the membrane proteins would be in the 10,000x g spin however every where else says the membrane proteins will come out with the ultracentrifugation (100,000 x g) spin. Was yours a mistake or is there a different prep step?

I dont know with how much material you started your extraction but expect to loose protein along the way, as a result sue a lot of starting material. Your vesicle should be in the 100 000g pellet

Dear Maddison,

the endosomes are, of course, membrane organelles. The "membranes" mentioned in the 100.000g pellet are not plasma membrane but endosomal membranes. Hope this makes sense to you.

Good luck with your experiments