You could try acidic glycine (0.1M). You could add some EDTA in this solution. The pH will be around 1 or 2.

The main problem will be to remove the glycine. You need to wash you exosome.

After the wash, you could lysate them in 2DE lysing buffer (urea, thiourea, CHAPS...)

The affinity procedure for isolating exosomes is a sufficient method but you could not prevent the contamination with the IgG heavy or light chains, especially using low pH buffers. The low pH or any further puffers to lose the antigen antibody complex is also sufficient to lose the antibody bead complex.

we use to ways to resolve the problem: 1) Performing the 2D gelelectrophoresis protocol without DTT. Then the light and heavy chains of the IgG would migrate in the gel at 50 kDa and at about 100 kDa and more (depending on the antibodies specificity) instead of 25 kDa and 50 kDa. Generally the 2D gels resolve proteins with molecular weights from 100 kDa - 5 kDa, depending on your gel conditions (sephadex and concentration of the gel). If you run the gels without DTT, it is possible to reduce the amount of the contamination because the high molecular weight proteins do not migrate into the gel matrix. Then it is possible to see more exosomal proteins instead of IgG proteins.

2) Crosslinking of the cd63 antibodies together with low pH or the further buffers (e.g. http://www.piercenet.com/product/binding-elution-buffers-igg-purification). Using this procedure only the exosome antibody complex is destroyed and the IgG are not eluated together with the exosomes. Unfortunately, the use of the cross linked antibody results in low rate of yield.

Good luck with your experiments.

Susanne

Hi, one additional alternative to consider is to covalently couple the primary Abs to the bead surface. This will at least reduce the amount of abs eluted off the beads together with the target. Dynabeads M-270 epoxy can be used as a solid surface for covalent coupling of prim Abs and used for capture of sub populations of exosomes.

kind regards

ketil

combining e.g. Dynabeads CD63/CD81/CD9 with a gentle elution protocol should ensure maximum exosome pull down and no heavy or light chain on the gel.

hi can be elute exosomes from dynabeads cd63 and use them on cell culture using glycine low pH......how to check the integrity and elution efficiency in such a case.

plz help

Hi,

We have succesfully eluted EVs from CD9, CD61, CD63, and CD81 monoclonal antibodies immobilized inside monolithic columns with pH 11.3 ammonium hydroxide or pH 11.3 carbonate bicarbonate solution. This elution strategy should work with antibodies on beads as well. The preparation of the solutions you can find in this article:

Article Fast isolation of highly specific population of platelet-der...

The eluate can be neutralized with HCl immediately after the elution to reduce exposure to high pH.

Br,

Evgen

Very nice reference by Fu et al NATURE COMMUNICATIONS | (2019) 10:4355

CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity.

Abs were covalently conjugated to Dynabeads and after exosome capture the vesicles were eluted off by using 0.1M sodium citrate/citric acid.

kind regards

ketil