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5. Protein Techniques

Protein Extraction


Protein Extraction

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in this video, we're gonna talk about the first step in purifying the protein, and that's protein extraction. So before we can even think about purifying the protein, we first have to be able to obtain our protein from a source, and that source of proteins are typically sells. So in order to obtain are proteins from cells. We have to perform protein extraction, and so the first step in purifying the protein as protein extraction. And that just entails the removal of all of the proteins from the source, which again are typically sells. And so the process of cell homogeneous ization is the process that involves the breaking open of cells in order to get them into smaller fragments and to release all of their contents into the solution. And so all of the contents of the cell are collectively referred to as the crude extract or the homogeneous. And so all the crude extract, or homogenous is is just the total released contents of the cell. And so the crude extract includes the organelles of the cells, such as the nucleus and a pleasant particular mitochondria, and the molecules of the cells, such as lipids, carbohydrates, nucleic acids and, of course, the proteins which we are interested in extracting. So what you can see is that protein extraction it's certainly not going to purify our protein, since we're gonna have a big mixture of all of these other things that were not really interested in. And so they're actually several different techniques that are used for cell homogenization or breaking open cells, and we're not gonna talk about the techniques and detail, but you should be familiar with this. A couple of different ways to perform sell homogenization, and the first is to use license buffers. And this is just a specific buffers or solutions that interact with the plasma membrane and break open the cell. Now the next way is to use a blender or a homogenize er toe physically break open the cells so literally these air instruments where you put the cells in them and you physically smash the cells open in order to relieve, get them to release their contents. And then the last way that we're gonna talk about is sonic ation and sonic Asian uses sound waves, toe break, open the cells and to perform sell homogenization. So in our example down below protein extraction, which will notice, is that we have our cell over here on the far left, and this is a red blood cell, and we're performing sell homogenization on this red blood cell so that we can get it to release all of its contents. So notice that the cell here is being burst it open, and it's releasing all of its contents into solution. And so we're able to extract the proteins in this fashion. So we also call it protein extraction because the proteins are being removed. But which will notice is that in addition to all of these proteins, so notice that we have these green proteins in here. We have thes blue proteins. We have black proteins. We have all these proteins. In addition to the proteins, we also have other structures and molecules. So notice that we have, um the nucleus is here. We have the end, a plasma, particular mitochondria. We also have RNA and DNA and carbohydrates. It's just a big mixture. And so this is together referred to as the crude extract, which again is all of the contents of the self. And so again, protein extraction does allow us to obtain all of our proteins, but it's certainly not a purified protein yet, and so you can see how we're going to need mawr techniques to continue to purify our protein. But the first step is to be able to obtain the proteins, and we have done that. So the next step in the process is differential. Centrifuge Gatien. Now, before we talk about that, we'll get a little bit of practice in our practice videos, so I'll see you guys there.

A microbiologist is looking to study a new version of beta-galactosidase (lacZ) from  E. coli cells. To purify lacZ for experimental studies, an effort is usually made to first: