You’ve cultured your cells and completed your treatments, now it’s time to harvest them and proceed to the downstream assays. Cell lysis is the crucial stage that determines if your experiment has a chance of producing the data that you have been waiting for. Part of the starting biological material is inevitably lost on each step of your experiment, so it’s critical to extract it all at the beginning. Often, the amount and quality of resulting biological molecules depend on the chosen lysis method.1
1. Frozen vs. Wet cells for Lysis
If you work with cytoplasmic enzymes or DNA, it probably doesn’t matter if you are going to do your cell lysis on “just-off-the centrifuge” cells or frozen cells. However, keep in mind that any method that involves wet, non-frozen cells does not completely stop biochemical reactions.2 If you need to get a snapshot of “normal” cellular activity, the sooner you freeze your cells and stop stressing the cells and thereby altering normal transcription and translation, the better.
After you decide on the starting state of your starting material, there are several things to consider when planning your lysis.
2. Cell Wall
You are lucky if you work with mammalian or insect cell lines, as they are only surrounded by a thin plasma membrane that doesn’t require mechanical effort to rupture. Gram-negative E. coli is a slightly bigger problem because of multiple rigid peptidoglycan layers. However, if you work with algae, fungi (that include yeast), archaea or plant cells, you need to get rid of at least parts of the cell wall, so cell lysis will require more effort than just putting in a detergent and heating cells up.
There’s a difference in cell wall thickness and composition within groups as well. Gram-positive bacteria have many more layers of the main cell wall component peptidoglycan than E. coli and it will require extra effort to lyse them. “Algae” is a diverse group with cell walls of various composition, so it’s worth checking recommended methods, if not for your species than at least for the algal family.
Significantly upscaling you prep without changing the lysis method often creates problems. For example, you were always doing your yeast preps for the western blot on a tabletop bead beater (for example) that takes eppendorf-size screw-top tubes. Now you need to do a large pull-down experiment where you need to lyse tens of grams of cells. It would be a mistake to just split your culture into smaller volumes. The smaller the volume, the more material you lose. But even leaving aside the time you will spend aliquoting and pooling you samples, it’s worth exploring lysis options for larger culture volumes: sonicator (suitable for medium-size preps), French press (not the coffee one) or cryogenic tissue grinder.
4. The Method for Lysis
You choose your lysis method depending on your downstream application. It can be either mechanical or enzymatic.
- Mechanical lysis methods include boiling cells with a detergent, vortexing with glass or ceramic beads, grinding cells in liquid nitrogen or sonication. Grinding with beads is suitable for isolation of soluble cytoplasmic proteins or the cell walls but not for preparation of intact mitochondria or plasma membranes. In these cases, enzymatic lysis or protoplast lysis are more appropriate.
- Enzymatic methods are based on using specific enzymes (no surprise there) to strip off cell wall and osmotic shock to release the cell content. This method is less harsh than the mechanical grinding, so suitable for isolating intact cell structures such as mitochondria.
- Detergent methods are quick, but you need to make sure that you know how to get rid of the detergent afterwards. The leftover detergent can interfere with your downstream biochemical assays.
Considering your lysis options before starting the experiment will save you time and reagents, and help you get the answers you were looking for the first time around!
- Sohrabi M. et al. (2016) The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology. J Microbiol Methods. 122:64-72. doi: 1016/j.mimet.2016.01.013.
- Lee S. J. et al. (2017) Cost-effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins, including phosphorylated eIF2?. Yeast. 34(9):371-382. doi: 1002/yea.3239.