Puromycin is an antibiotic derived from the bacterium Streptomyces alboniger, widely used in molecular biology and cell culture research. It serves as a valuable tool in laboratory settings, particularly where selective pressure on cells is required.
How Puromycin Works
Puromycin functions by interfering with protein synthesis, acting as an amino-nucleoside antibiotic. It mimics the 3′ end of an aminoacyl-tRNA molecule, which is typically responsible for delivering amino acids to the ribosome during protein production. This mimicry causes puromycin to be incorporated into the growing polypeptide chain, leading to its premature termination.
The incorporation of puromycin results in the release of truncated, non-functional proteins, which ultimately induces cell death. This mechanism is effective in both prokaryotic and eukaryotic cells, making puromycin versatile in research applications. Its rapid action can lead to cell death at relatively low concentrations, sometimes killing over 99% of non-resistant cells within a day.
Key Applications in Biological Research
A primary application of puromycin is in applying selective pressure to establish stable cell lines, a process commonly used in genetic engineering. Cells are modified to express a puromycin resistance gene, such as the pac gene. This gene encodes puromycin N-acetyl-transferase (PAC), an enzyme that inactivates puromycin by acetylation, thereby conferring resistance.
Once cells have been engineered with the resistance gene, puromycin is introduced into the cell culture medium. Only cells successfully expressing the resistance gene survive and proliferate in the presence of the antibiotic. Non-resistant cells are quickly eliminated, often detaching from the culture plate, allowing researchers to isolate the desired genetically modified cells. This selection process is widely employed in techniques like gene editing using CRISPR/Cas9 systems, RNA interference (RNAi) studies to silence specific genes, and the general generation of stable cell lines for diverse research purposes, including the production of specific proteins or studying gene function.
Important Considerations for Use
Proper handling and storage of puromycin are important for maintaining its stability and ensuring accurate experimental results. Lyophilized puromycin powder should be stored at -20°C for long-term stability, while solutions can be stored at 4°C for short periods or -20°C for up to one year, avoiding repeated freeze-thaw cycles.
Given puromycin’s cytotoxic nature, strict safety precautions are necessary during its handling. Researchers should always wear personal protective equipment, including gloves and eye protection, and work in a biosafety cabinet or fume hood to prevent inhalation or skin contact. Determining the optimal working concentration for different cell types is also important, as concentrations can vary (e.g., 1-10 µg/mL for mammalian cells, depending on sensitivity and desired selection stringency). An initial experiment to titrate the antibiotic is recommended to find the lowest effective concentration that kills host cells without excessive toxicity to resistant cells.