Greetings from the Department of Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee, where I’m ensconced in the lab of Dr. Louise Rollins-Smith. After years of studying genes encoding antimicrobial peptides, as of a week ago I still had yet to handle an actual antimicrobial peptide, so I am here learning how to work with them. Today I learned how to extract peptides from frogs. You give the frog a shot of norepinephrine, a stressor hormone that induces the frog to release the contents of its skin glands. You place the frog in a beaker of buffer for about fifteen minutes, and the frog secretes its antimicrobial peptides into the buffer. You add some acid to denature any enzymes that might break down the peptides, and you’re good to go. Other researchers use electroshock methods to induce peptide secretion, but that sounds painful. Alternatively, if you know the sequence in advance, small peptides can be synthesized artificially.
I’m also learning how to assay the antimicrobial activities of a peptide. You add known concentrations of sterile peptide to known concentrations of bacterial or fungal cells (for fungi, you can actually count the cells under a microscope to calculate how many you have). You incubate them for a day or a week, depending on the microbe being tested. At the end, you assess microbial growth by how cloudy the solution is, since cells will block the light (using a spectrophotometer, of course, not just your naked eyes). Keeping everything sterile takes some effort (working under a hood whenever solutions are exposed to the air, passing solutions through filters, using lots of sealed and disposal tubes and tips, etc.). I’m accustomed to keeping an area free of PCR product, but keeping an area free of microorganisms is slightly different. It also turns out that antimicrobial peptides don’t always want to go into solution and stay there, which in practice can be a bigger issue than I ever would have imagined.
In general, there is still a sizeable gap between evolutionary geneticists and protein biochemists. The former group is good at identifying nucleotide substitutions that have been favored by natural selection, and the latter group is good at assessing the functional differences among protein isoforms, but only recently have the two fields started to merge. We still have a lot to learn about how non-neutral genetic differences identified by statistical tests of DNA sequence data translate into differences in protein function.