I am struggling with the method for subcellular membrane fractioning.
I use Optiprep and tried continuous and discontinous gradients 1-25% and 10-40% with human melanoma cells. The method was run for 3-18h with 49.000xg - 200.000xg.
I do get the plasma membrane but ER and Golgi seem to stick together.
Does anyone know how I could seperate the last two? Or what else I could try?
I already tried some things from the Axis-Shield protocols but it seems I dont find the right conditions for my cells.
Thanks for helping!
Hi, Bettina,
As for my experience, It's really hard task to separate ER from Golgi.I did not clearly understand if you tried to separate ER+GA fraction first and then repeat centrifugation with this only fraction under various conditions?
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Hej Maria,
My procedure is like this:
+ Homogenize cells (Miltenyi gentleMACS Dissociator)
+ 1.000xg 10 min take Post nuclear SN
+ 100.000xg 40 min pellet membrane
+ homogenize membrane pellet and transfer to gradient
I tied loading on top and at the bottom. Best case would be to separate plasmamembrane, ER and Golgi in one run.
I separated alot of mitochondria with a discontiuous percoll gradient.
I never did ER but one thing to try if you havent already is Make sure the temperature of the centrifuge is 4 degrees C or lower. any warmer my separations never worked.
In general , I am obsessive about the pH of the solutions in the gradient. I have noticed that for mitochondria , 6.8 to 7 works best. The ER , golgi separtion might have a optimal pH as well. You probably tried these simple changes already but thought I would mention it anyway .
I agree with Michael Joseph in general. I guess it may work. Though mitochondria are usually much easier to separate than ER and GA fractions.
As for separating PM, ER and GA in one run...I'm not sure. I work with fungal cells for which it seems to be completely impossible (I mean most of all ER and GA). Anyway, I guess with the human cells it is not so bad because, as I remember, they GA of such cells is quite abundant unlike fungal cells.
Maybe, along with suggested above, the change of gradiend medium will help?
1. Once you remove the nuclei try to remove the mitochondria with centrifugation at 10,000g for 10 mins (you may have to adjust it), and that should help you to get rid of mitochondria.
2. Wash with Na2CO3 will help you to remove the cystosolic proteins
3. Remaing shoud be mambrane from organelles and plasma
4. Then follow the gradient approach to separate those membrane stepwise.
I hope this might help you.
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