by Macy Osborne-Frazier
Fun Rating: 4/5
Difficulty Rating: 2/5
What is the general purpose?
Isolating protein from cells allows a scientist to use the cells grown in cell culture to determine how much of a protein of interest is present, known as its expression level. By isolating proteins from cells, scientists can learn valuable information about how a protein’s expression levels can be changed by different experimental conditions.
Why do we use it?
Often scientists are interested in expression levels of a certain protein in the context of various diseases. Certain genes, like oncogenes, encode proteins that are very important for the development of cancer or other diseases. In addition, some diseases or cancers cause cells to stop making proteins that are very important for healthy functions of the cell. Scientists can use cell culture to grow many different types of cells to understand how different diseases or drug treatments affect the proteins important for healthy cells. Understanding how diseases and drugs change the amount of protein in cells can help scientists determine better ways to treat or cure diseases.
How does it work?
As mentioned, scientists often isolate proteins from cells grown in cell culture. First, cells are grown until they cover the surface of a petri dish and are then collected for protein isolation. The exact process of isolating proteins may change depending on the protein of interest and starting material, but all procedures follow the same general outline: lysis, isolation, and quantification.
Lysis
Proteins are present all throughout cells, from the cell membrane to the nucleus. The first step when isolating protein is to break open, or lyse, the cells so the proteins are accessible for purification. Cells can be lysed using several methods, but most often the cells are either lysed by chemical or mechanical methods. Chemical methods use a chemical solution to break the cell membranes. Mechanical methods physically break cell membranes by force. Once the cell membrane is broken, the proteins can be separated from the solution.
Image: Illustration showing types of cell lysis (Created by author with BioRender.com)
Isolation
After the cells of choice are ruptured by lysis, the proteins in the cell must be isolated from the other cellular components such as DNA, RNA, and organelles. Proteins are a different size and weight than other cellular components. Therefore, scientists can isolate proteins from the solution containing ruptured cells by centrifuge. A centrifuge spins the solution of ruptured cells at a high speed to use the force of gravity to isolate different parts of a solution. By spinning the cellular solution at a specific force, a scientist can isolate the proteins from the solution. The proteins will form a pellet at the bottom of the tube, and the other cellular components will stay in the liquid part of the cellular solution, called the suspension. The liquid solution containing the unwanted cellular materials can then be disposed. The protein pellet can be stored at negative 80 °C until it is quantified to be used for an experiment.
Image: Illustration showing centrifugation of lysed cells (Created by author with BioRender.com)
Quantification
After isolating protein, scientists likely need to know how much protein is present in the pellet before they continue any experiments using the pellet. To quantify the amount of the protein, scientists can use a Bradford assay. After quantifying the amount of protein, a scientist can conduct a Western blot to look at expression of specific proteins.
