Laura Lane has worked as a health and science journalist since 1997. She received her master's degree in biology from Stanford University. Since then, she has written for the Dallas Morning News, the Contra Costa Times, Shape magazine, WebMD, Yoga Journal, Diagnostic Imaging, the International Medical News Group, The Scientist, Bio IT World and Biocompare.
When it comes to protein extraction, it’s all about balance: You need a method strong enough to crack open the cell wall and/or membrane, yet gentle enough to protect the vulnerable protein molecules inside.
It’s a tall order that requires months of optimizing and a good dose of creativity. Although the protocol you formulate must maintain the integrity of the protein, it must also take into consideration the downstream application, such as mass spectrometry or binding studies, for the extracted proteins. The two goals often run counter to each other.
To make sure the proteins are fit and ready and in sufficient quantities, your extraction technique requires a fine balance of reagents, temperatures and procedures. Formulate your recipe by choosing from decades-old physical methods as well as newer detergent- and enzyme-based approaches.
Let’s get physical
Researchers adopting physical methods are breaking through cell walls and membranes much as medieval conquerors broke into fortresses: with brute force. Blenders use mechanical force to grind up cells and tissues. Some blenders use blades and rotors, mostly for industrial-size sample volumes. In the laboratory, blenders usually operate by agitating tiny beads to physically break open the cell.
Homogenizers rely on pressure to create a shear force that breaks open cell membranes. Sonicators use sound waves to blow open the cell membrane. Repeated cycles of freezing and thawing create ice crystals that weaken the cell membrane. The mortar and pestle come in handy for grinding up plant cells, with their hardy cellulose walls, such that the membranes are exposed for lysis.
“It’s definitely a pain,” says Aman Husbands, a postdoctoral fellow at Cold Spring Harbor Laboratory in Cold Spring Harbor, N.Y., describing his work with plant cells, which require an additional step of dissolving cell walls before lysing cell membranes.
Husbands isn’t so much complaining about the time and labor involved; he’s mainly referring to protein stability. The more time given to extracting proteins, the lower the quality of those proteins. But to improve yield, he must spend time taking an extra step—a consideration Husbands must weigh in designing his experiment.
First, Husbands freezes the plant material, usually maize or Arabidopsis, with liquid nitrogen. He grinds it all up with a mortar and pestle. At this point, he could extract some protein. But, to collect meaningful amounts of the target protein, he relies on a sonicator, which he says is key to extracting his target protein.
“The longer you have protein sitting in buffer and not in native conditions, the more [protein] you lose,” he says. “The faster you do it is better.”
Enzymatic, automatic, systematic
Yeast and bacteria also call for extra time because of their hard outer layers. Many researchers address that hurdle by sonicating and homogenizing. One downside of this approach is the cost of the equipment. The smallest sonicators start at nearly $2,000 to handle up to 50 ml of sample, and the prices go up from there. Also, the agitation and friction generate heat that can compromise proteins.
Although some researchers chill the equipment and keep samples on ice, others circumvent the problem by using enzymes to do the job. Lysozyme breaks down the peptidoglycan layer of the bacterial cell wall. Zymolase and lyticase are most popular for tackling the yeast cell wall, and cellulases digest cellulose to dissolve plant-cell walls.
Detergent for the delicate cycle
Cells without walls, such as mammalian and insect cells, require only detergents for lysis. By interfering with the phospholipid bilayer, amphipathic detergents take apart the membrane, dissociating the interactions between and among lipids and proteins.
Detergents come in several forms: denaturing, nondenaturing, anionic, ionic and combinations thereof. Some detergents are mild, and some are more stringent. You can purchase detergents created to lyse specific cells, such as animal, bacterial, yeast, insect and plant.
Along with detergents, successful cell lysis and protein extraction requires aiding-and-abetting reagents: buffers, salts, reducing and alkylating agents and protease and phosphatase inhibitors. Trial-and-error experiments with different concentrations of all the players result in the optimal mix for protein extraction.
You’ll always find the need to do some optimizing, but kits and prepared solutions marketed for specific types of cells, proteins or applications might save you some time. For example, a kit created to extract proteins from bacteria will include DNase I to remove sticky genetic material, which you don’t encounter with eukaryotic cell lysis unless you also break open the nucleus.
Extracting protein and optimizing “can be messy,” much like starting construction with a handful of “nuts and bolts,” explains Ryan Austin, a postdoctoral fellow at the Institute for Systems Biology in Seattle, who is studying protein biomarkers for prostate cancer. “You start with a method that works and then use baby steps from there.”
In his research, Austin needs to extract protein from yeast. Like everyone else, he’s tinkered with cell lysis methods to maximize the yield -- passing lysates through the French Press a third time, for instance. But his chief way of collecting enough protein to study? “Cranking up the sample volume,” he says. “Whatever works.”
Going the distance
Extracting a single protein is difficult enough. Going after multiprotein complexes spells challenge. Why do it? Because proteins very often function in conjunction with others, says Mark Gillespie, also a postdoctoral fellow at the Institute for Systems Biology, whose research involves looking at transcription factor complexes.
However, “to identify those proteins is different,” Gillespie says. “You identify them in a dynamic matter with more time points and measurements.”
And to extract them in their native state, associated with other proteins, requires juggling, particularly if they lie in the nucleus. On the one hand, you use high salt concentrations to dissolve the nuclear membrane knowing that such conditions cause proteins to dissociate from each other. Gillespie dialyzes the salt out to preserve the complexes, but this step greatly reduces his yield. “It’s not very easy,” he says.
Then again, no one ever said protein extraction would be. But with lots of fine-tuning, and a little imagination, the ultimate protein extraction should be within your reach.