Gibco Blasticidin S HCl is a potent antibiotic used in molecular biology laboratories. Derived from the bacterium Streptomyces griceochromogenes, its primary role is as a selection agent in cell culture experiments. Scientists use it to isolate cells that have been successfully altered with a genetic vector carrying a blasticidin resistance gene. This process ensures that only the genetically modified cells survive and grow in the culture.
Mechanism of Action
Blasticidin functions by halting protein production, a process known as translation, in both prokaryotic and eukaryotic cells. It targets the ribosome, the cellular machinery responsible for building proteins. The antibiotic binds to the peptidyl transferase center on the large ribosomal subunit. This action obstructs the formation of peptide bonds, which link amino acids into a protein chain, thereby stopping protein synthesis.
For a cell to survive exposure, it must possess a resistance gene, most commonly bsd or bsr, introduced along with the gene of interest. These genes produce the enzyme blasticidin S deaminase. This enzyme chemically modifies the blasticidin molecule by removing an amine group, a reaction known as deamination. This alteration creates an inactive form, deaminohydroxyblasticidin S, which can no longer bind to the ribosome, allowing the cell to continue producing proteins.
Experimental Protocol and Kill Curve
The effective concentration of blasticidin depends on the cell type and must be determined experimentally for each new cell line. This process involves creating a “kill curve” to find the lowest dose that eliminates all non-resistant cells over a set period. The procedure begins by plating the parental, non-modified cells in a multi-well plate at a moderate density, allowing them to adhere overnight.
The following day, the growth medium is replaced with fresh medium containing a range of blasticidin concentrations, from 1 to 20 µg/mL. A control group is maintained in a medium with no antibiotic to serve as a baseline for normal growth. The cultures are then incubated, with the selective medium being refreshed every three to four days to maintain the antibiotic’s potency.
Over a period of 7 to 14 days, researchers monitor the plates daily to observe the rate of cell death at each concentration. The objective is to identify the minimum concentration that results in complete cell death for the non-resistant population. This value is then used for subsequent experiments to select for cells that have successfully incorporated the resistance gene.
Recommended Selection Concentrations
While a kill curve is necessary for optimizing selection, established concentration ranges provide a useful starting point for many common cell lines. These published values can help guide the design of the kill curve experiment. For example, some frequently used cell lines have the following working ranges:
- Human Embryonic Kidney 293 (HEK293) cells: 2 to 10 µg/mL
- HeLa cells: 2 to 10 µg/mL
- Chinese Hamster Ovary (CHO) cells: 5 to 10 µg/mL
- A549 human lung carcinoma cells: 2.5 to 10 µg/mL
- NIH/3T3 mouse fibroblast cells: around 400 µg/ml
These are guideline concentrations. Factors like cell density, passage number, and media formulation can influence a cell line’s sensitivity, reinforcing the need to validate the optimal concentration for your unique experimental setup.
Handling and Storage
Proper handling and storage of Gibco Blasticidin are important for maintaining its activity and ensuring experimental reproducibility. The product is supplied as a sterile-filtered liquid solution at a concentration of 10 mg/mL in a buffered solution. For long-term storage, the stock solution must be stored at -20°C and protected from light. When stored under these conditions, the product is stable for at least nine months from the date of manufacture.
For short-term use, an aliquot of the stock solution can be kept at 4°C for up to two weeks. It is recommended to create single-use aliquots to avoid repeated freeze-thaw cycles, which can degrade the antibiotic. Once diluted into cell culture media, the blasticidin solution remains stable for one to two weeks when incubated at 37°C. When working with the concentrated solution, standard laboratory safety precautions, including wearing gloves and safety glasses, should always be followed.