Hi,
I'm using density gradients to isolate exosomes! I've read that they appear in the 1.11g/mL fraction. Following in the steps of a few authors, I created a self-generating density gradient by using a transfer pipette to layer on 40%, 20%, 10%, and 5% mixtures of OptiPrep with sucrose/tris.
I calculated out the right time and rpms to mimic their methods and based on the concentrated media overlaid on top, it seems like there are multiple gradients.
My first problem is that I have no idea what fraction is what. I've read that you can use a spectrophotometer to determine concentration through absorbance at 244nm. I tried this together with another paper's assertion that the exosomes manifest in fractions 6 and 7, counting down from the top.
The thing is, my reads are all over the place. I think it's because I took the fractions wrong. I can't find any sort of protocol on this, either. Is there some technique to harvesting the right gradient without getting others?
Hi Colin
I have used CsCl gradient before at 1.35 g/ml loading density. After ultracentrifugation I have measured weight of each ml of solution (fraction) in the tube seperately, that's how I get what is the density (g/ml) of each fraction. Then you can test whether at 1.11 g/ml, you have the exosomes or not. Hope that helps.
Hi Megha,
Weighing might be an option. But is there a good technique to harvest these fractions? I'm worried I'm taking up several different gradients at once, since the liquid in my pipette tip becomes heterogeneous. It's a tiny tube, a little more than 12ml and this method of isolation is supposed to yield around 12 distinct fractions. The problem is, they're very hard for me to see after the three media-colored fractions are removed. I can see the gradients when they're agitated, but I feel like that results in contamination of the gradients since they no longer appear homogeneous. Should I use a needle? A serological pipette?
I've found that there's a way to measure out the fractions without using a mock gradient with just a plate reader. But I'm honestly guessing during my harvesting of either the mock or the real samples. All I'm doing at the moment is counting 12mls. I'd like something that gives me a bit more certainty. Apparently even 2% change in Iodixanol results in 0.015 difference in density.
Hi Colin,
When I collect fractions, I use a 1 ml Pipettor, slowly turn the tube and make sure the pipette tip only touches the surface of the liquid. But remember you gradually need to lower the tip so you don't pipette air. In this way you will pipette only 1 ml of fraction without disturbing the others. Also when you handle the tubes after centrifugation, handle very gently to maintain the gradient. I am using a 12.5 ml tube as well and seperating 12 different fractions with density increasing from top to bottom. As far as measuring the density and extracting each fractions carefully, this method works efficiently. You just need a bit of practice to extract by touching only the surface and rotating the tube slowly for the fraction.
How do you guys convert the absorbances into densities of fractions? Is there a formula?
Hi Camila,
Yup! Here you go! http://www.axis-shield-density-gradient-media.com/S09.pdf
In a nutshell: Put it in a plate reader, read absorbance at 340. Match up the table to the readout to get the density. It's a bit tedious, but if you're good with Excel, I'm sure you can make some kind of command for it.
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