Thank you

that was a good reference

Hi Andrei,

That is a good reference. So this implies detection of GAPDH in the otherwise purely isolated mitochondria is a normal phenomenon. Then I can get on with my work confidently.

Thank you.

Hi,

Thanks for the tip.

Hello Emma,

if you have a contaminations with cells, you can do a low speed centrifugation step (~ 600 g) and then apply the supernatent to the MACS beads.

Cheers,

Stefan

You may check this paper to see how to isolate mitochondria, which have been magnetically tagged in the cell. GAPDH and other glycolitic enzymes are always there probably because they act as a complex on both sides of mitchondria membrane .

http://onlinelibrary.wiley.com/doi/10.1002/anie.200800357/pdf

I do n't know about MACS. I usually isolate by dounce homogenizer. Mitochondria is small and fragile organ. Try to handle cells in a delicate way and always use ice.

Isolation of mitochondria and only mitochondria can be difficult. Perhaps an affinity column approach as described on page 185 of "Determination of the Orientation of Membrane Vesicles Derived from Mitochondria. H.J. Harmon. Journal of Bioenergetics and Biomembranes 19, 167 189,1987" would help. Here, the column is loaded with cyt c and the mitochondria bind to the cytochrome c. The mito can be eluted easily using a wash with 0.15 M KCl in buffer. There are other easy centrifugation tricks (1-step using a Sorvall or microfuge) that can be used with polylysine or protamine sulfate (very cheap) as well. Coat the mag beads with cyt c (not all cyt c's are the same!) For this, use Sigma Type III.

You can do it without a column; coat the beads or polystyrene or Sepharose beads with cyt and then spin them down (mito are attached); elute with 0.15 M KCl.

Have encountered this before. We wanted to isolate pure intact mitochondria of normal (not inverted) orientation and have used this and protamine sedimentation tricks to get them for chemiosmotic studies as well as membrane protein location studies. Cyt c, protamine, polylysine (more $$$$) all will bind at the exposed cyt c site and allow you to separate;; antibodies work also, but too $$$$$$$$. Hope this helps.

The following study used immunoblotting to confirm the separation of mitochondria from nuclear preparations, by detecting PSP1 and MnSOD. Might be something to try alternatively.

http://www.ncbi.nlm.nih.gov/pubmed/21854988

I want to thank everyone for their contributions. Gathering from your ideas, I think the best option will be to try another marker to confirm separation of mitochondria from nuclear preparations as some people have already indicated.

Thank you

Hi Emma,

Have you observed high yield of mitochondria using MACS kit? I am now also using this kit in my thesis but I had low yield of mitochondria to perform RNA isolation.

Hi Elvan,

To obatain enough mitochondria for RNA isolation, I think you require large cell numbers. You can check the protocol for isoaltion of mitochondria by MACS kit using large number of cells. http://www.sciencedirect.com/science/article/pii/S0003269709001468

Regards

Thanks a lot Emma

Regards