What are the techniques used to extract protein through cell lysis?
Before a protein sample can be isolated and identified by electrophoresis and Western blotting, the proteins must be separated from component cell parts. This means cell walls and membranes need to be broken apart, or lysed. But lysing techniques should be chosen carefully, since overly aggressive activity can degrade the proteins and make genetic samples difficult to reproduce. Below are a few of the most common and trusted cell lysis methods.
Sonication involves inserting a tip into a test tube or eppendorf and sending a high intensity sound wave throughout the sample to disrupt cell walls and shatter tissues. Sonication is easy, and it can be applied to samples of almost any size, but it should be used with care, since this method may cause proteins to overheat and denature (breakdown). We recommend using multiple, short sonic bursts as opposed to one long burst. All sonications should be performed on ice and the samples allowed to rest and chill between bursts.
Liquid Nitrogen Grinding
Using this method, researchers blend protein samples with liquid nitrogen until they freeze and become brittle. The samples can then be ground with a mortar and pestle. Grinding involves low risk to the integrity of the proteins, but liquid nitrogen can be dangerous. Grinding in liquid nitrogen is a great technique for difficult samples, such as plant and fungi samples.
Bead Beaters
This technique involves combining a cell sample with tiny beads in a bowl, which is then mechanically agitated in order to break the cells apart. To avoid overheating the sample, this should be done in a cold environment and the sample should be allowed to rest and cool between beating cycles.
Buffers
The simplest, but also the most expensive way to lyse cells and tissues is by using a specific lysis buffer. The lysis buffers are designed to break open cells and tissues chemically. A large selection of buffers are available ranging from mild lysis buffers to strong, chaotropic, denaturing buffers. The lysis buffers use a combination of buffering agents, salts, detergents, reducing agents, chaotropes and lytic enzymes to release proteins. The use of chemical lysis can also be combined with the above mechanical lysis techniques to improve extraction of proteins.
For a review of the lysis buffers available from G-Biosciences, including non denaturing, denaturing, RIPA and inclusion body lysis buffers, download our Sample Preparation Handbook.
Important!
Don’t forget that with all these techniques designed to release your protein of interest, the endogenous cellular proteases are also released. So don’t forget to ALWAYS use a Protease Inhibitor Cocktail. Check back for more information on protease inhibition!