Cell fractionation-help! - (Oct/31/2007 )

I used a dounce homogenizer to lyse my cells (roughly 20 million, resuspended in 0.8 ml buffer, 20 strokes with the tight fitting pestle). Then I centrifuge the lysate at 1000g, collect the supernatant then spin at 6000g to separate mitochondrial to cytosolic fractions. Then I analysed my protein of interest by WB and find the expression level roughly comparable in both fraction treatment VS control. However, this is the problem. When I look at whole cells RIPA lysate the expression level is VERY different between control and treatment. So where did the difference go? The protein is not a nuclear protein.

If anyone can see where the "missing" difference went I would most grateful.

Did you measure protien conc. of the samples before verifying in western and load equal amounts.

Yes, the loading control confirms equal loading

Did you check to see if the homogenization stage works well (as in, cells were broken efficiently)?
Which fractions are you checking? The supernatant (almost total cell lysate after the 6000g spin or the pellet of that step or both? Is your protein membrane bound or at least sometimes associated with the membrane? One main difference I can think of according to your description is the detergents in RIPA buffer, which would solubilize membrane proteins. Your 6000g spin certainly would not pellet all the membrane fractions, but it is possible that some big membranes could be spinned down. If you are checking the pellet and found no difference, then the interested protein is still in the supernatant. If you are checking the sup instead, protein could be in the big membrane that was pulled down in the pellet. Did your homogenization buffer containing sucrose? That could help a little to avoid the cross-contamination in between fractions. Do you really need the 6000g step? If not, skip it and try again.

Thanks. After 6000g I check both the cytosolic (supernatant) and the mitochondrial (pellet) fractions. As I described, both fraction contains roughly comparable level of the protein. Although I know that there SHOULD be a quite a big difference. Does anyone know what is in the pellet after the 1000 g spin apart from nuclei and unbroken cells? And Yes, the lysis buffer does contain sucrose.

Try 100,000g 2hrs ?

I am not sure about what you expected to see, so here are two possibilities (remember that this applies only if the bad results is caused by unefficient fractionation, i.e. even control proteins are not found at expected fractions):

1. If you want to see protein in the pellet but still found them in supernatant, then you need to either spin for longer time or at higher speed
2. If you want to see protein in the supernatant instead, then it is the opposite way.

Since you said that when you use RIPA buffer you see big difference, I suppose that meant you see more protein in the supernatant? If so, it is because the detergent in the buffer has solubilized the membrane-bound proteins --> it is in supernatant. In that case, it is not necessary to mean that the protein is not in mitochondria.

In either cases, bear it in mind that you need proper controls to make sure that the fractionation is good. Say if you want the protein in the mitochondria, thus the pellet fraction so you spin very long and at very high speed then of course all the membrane bound compartment will be pulled down. But that did not mean that your protein is mitochodrial protein, since it could, in this instance, be at ER, Golgi, endosomes... This really depends on the purpose of your experiment to choose a method of fractination that would work best and show what you mean. If you just want to separate membrane-bound proteins from cytosolic proteins, sure use high speed. But if you want to do more delicate fractionation, e.g. mitochondria from other organelles, this would need much more care. I have never worked with mitochodrial proteins, so I would have to suggest that you double check the published papers. There will have much more detailed methods that could help you.

By the way, the supernatant after the 6000g spin is NOT cytosol. Cytosol means the water-soluble fraction of the cytoplasm, after all the organelles have been isolated. 6000g spin do not separate all organelles. There are plenty membrane-bound compartments still in the supernatant at this speed.