I want to obtain purest mitochondria and MAMs from T cells.

For crude mitochondria isolation (differential centrifugation) I've used Abcam's benchtop mitochondria isolation kit. However, for percoll gradeint protocol the following was used:

Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Mariusz R Wieckowski1,4, Carlotta Giorgi2–4, Magdalena Lebiedzinska1, Jerzy Duszynski1 & Paolo Pinton

But I've obtained one band at the first gradient interface.

So what I did is I increased the time of centrifugation (first ultracentrifugation step) to 1 hour instead of 30 minutes (at 95000g).

I have obtained a very faint band at the bottom of the polyallomer ultracentrifuge tube but it is still MAM contaminated (i.e. mitochondrial pellet is calnexin positive an ER/MAM marker).

NB: Starting amount of cells is 200,000,000.