Hi,
I have a question regarding the parameters (speed and length) during sucrose gradient centrifugation for polysome profiling.
I'm attaching one typical chromatogram from my experiments. The monosome peaks (40S, 60S and 80S) are very well separated but the polysome peaks are pretty close to each other.
I would want to have a better separation of the polysome peaks and I actually want the monosome peaks separated less (right now they take up almost half of the gradient). I'm thinking about maybe centrifuging less would achieve my purpose, either by decreasing the centrifugation speed or length. Would that work?
Right now I'm using the following parameters: 10%-50% sucrose gradient; SW55 rotor; Centrifuge at 47,000rpm for 1h.
Does anyone have any empirical experiences about the parameters that would separate polysomes well?
Thank you!
Zhizhou
Hello, unfortunately I haven't worked with sucrose gradient partitioning. But trying with less speed sounds pretty logical to me.
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