![]() This comes from knowing your protein (see above). I know from personal experience that the insulin responsive glucose transporter, GLUT4, is a very grumpy protein. Transmembrane proteins can be very, shall we say, temperamental when it comes to purification. You also need to know the characteristics of your protein, as its function could also play a part in choosing your method. There are different types of fractionation (which we’ll get to later), and the type you choose will depend on where your protein is likely to be, and what you want to do with it. If you just want to look at a protein in isolation, consider a method like immunoprecipitation instead.įrom the outset, the first thing you need to know about your protein when planning your fractionation experiment is where you expect it to be. There are plenty of reasons to carry out fractionation you may want to look at protein translocation, membrane recruitment and sequestration, or as mentioned above investigate a single process without other cell functions interfering. One of the first things this knowledge will help you to identify is whether or not fractionation is even necessary. This phrase is going to come up a lot, but don’t worry if you forget it: I will be repeating it. 1. Know your proteinįirstly, and most importantly, know your protein. Here are my tips for successful subcellular fractionation. It is useful for looking at a single type of organelle in isolation, and allows processes to be studied in a cell free environment, without interference. High concentrations of sucrose are used to separate cell fractions based on their density. Subcellular fractionation is a method that dissects cells into their various organelles.
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