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How to detach / elute Exosomes from CD-63 coated beads

I´m trying to performe an 2D elektrophroesis with exsomes istolated with CD63 postive beads.

We first perfromed an 1D with silver staining. We lysed exosomes bound to beads with SDS buffer. Silver staining showed a huge band at 50-55 kD and one at 20 kD. We speculate that this might be the heavy and light chains of the CD-63 antibody.

In the 2D this immunoglobulins might disturbe analysis.

Does anybody know a how to elute exosomes from beads without contamination of CD63 IgG? Is there any buffer which can be used?

Thank you for your help!

Lucian
asked May 14, 2014 in ExosomesTalk by Lucian Beer

2 Answers

Ketil or Axl would be the best to advise on that. Its challenging of course- if you want to elute fully intact exosomes, after they were captured with Abs on the surface of magnetic beads. Either Abs or capturing moieties should have a reduced affinity (so that its relatively easy to disrupt the interaction), or Abs can have cleavable spacers...
answered May 19, 2014 by Vlassosv
Hi,

In terms of avoiding heavy and light chain contamination I would suggest trying e.g. anti-mouse IgG HRP  Trueblot from Rockland. works very well and will not recognize neither heavy or light chain of the Abs eluted off the beads.

kind regards

ketil
answered May 20, 2014 by ketil.pedersen
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