Can anyone recommend a method to isolate early endosomes from animal tissue without using ultracentrifugation?
I think that you could try to use immunoisolation, a powerful tecnique for the isolation of cells or of subcellular organelles. Indeed, you could use antibodies against early endosomal markers (EEA1, Rab5, Rab4,etc.) coupled to agarose resin or other kind of resin or magnetic beads, in order to separate early endosomes from other cellular components. I know that immunoisolation of organelles using magnetic supports has been successfully used to purify, for instance, peroxisomes. As you use beads to select your organelles you need either a magnetic support (if you use magnetic beads) or centrifugation (but no ultracentrifugation). Clearly, you need good antibodies.
Hi,
In general, all membrane bound organelles can be isolated using the Dynabeads. In combination with the your own organelle specific antibody the immuno-magnetic isolation approach has several advantages:
• The method is rapid and gentle
• Does not require any labour-intensive density gradient and ultra-centrifugation.
• Organelle populations with different density can be isolated.
• Highly pure organelles are obtained.
.
In addition to the functionalized beads a magnet is required.
. You can choose between two techniques:
- Direct technique: coat the Dynabeads® with your antibody/ligand by incubating the two together for a short incubation period. The coated beads are then mixed with your crude fraction. Target organelles bound to the Dynabeads® are separated by the use of a magnet and washing is performed in an equally fast and easy manner.
- Indirect technique: mix the antibody/ligand with the target containing crude fraction and incubate to let them bind. Then add the Dynabeads® to the sample, incubate for a short period to let the target-antibody/ligand complex bind. Separate and wash using a magnet. The indirect technique can not be used if the target specific antibody/ligand is to be coated directly onto one of the surface activated beads.
Due to the speed and efficiency of isolation, the direct technique is recommended. However if the antigen on the target organelles is inaccessible (e.g. buried within the vesicle), the indirect technique may be advantageous.
Some references for isolation of endosomes and lysosomes.
• R. Gagescu, N. Demaurex, R. G. Parton, W. Hunziker, L. A. Huber, and J. Gruenberg. The recycling of endosome of madin-darby canine kidney cells is a mildly acidic compartment rich in raft components. MOLECULAR BIOLOGY OF THE CELL 11:2775-2791, 2000.
• C. J. Provoda, M. T. Waring, and K. M. Buckley. Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis. Journal of Biological Chemistry 275 (10):7004-7012, 2000.
• T. E. Tjelle, B. Saigal, M. Froystad, and T. Berg. Degradation of phagosomal components in late endocytic organelles. J.Cell Sci. 111:141-148, 1998.
• N. Vitale, K. Horiba, V. J. Ferrans, J. Moss, and M Vaughan. Localization of ADP-ribosylation factor domain protein (ARD1) in lysosomes and Golgi apparatus. Proc.Natl.Acad.Sci.USA 95:8613-8618, 1998.
kind regards
ketil
Hi Ketil,
thanks for your detailed question, I will go through the literature
Clara
Hi Clara, thats great. Feel free to contact me at [email protected] as well. ketil
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