This question is regarding the sucrose denisty centrifugation that is used during polysomal fractioning (polysome profiling technique). I was wondering, what is the difference between using let's say 15%-45% sucrose concentration than 10-30%? Does anyone have any idea how may this affect the outcome?
Polysomes are high molecular weight complex of mRNA and ribosome. In a process to separate them we require wide range of % of sucrose concentration, hence in most of the widely used protocols a range of 5-50% sucrose concentration is used so that a layer of six sucrose gradient is obtained. With a range you have queried 15-45% or 10-30%, there will be inefficient separation of polysome. Ultimate outcome is you will miss out on individual factors that play a role in translational machinery, if translation is what you eventually intend to study.
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