|G264||1.0 ml; 10 mg/ml|
|Description||Extracted from Streptomyces alboniger bacteria, Puromycin is an aminonucleoside antibiotic that inhibits protein synthesis by interfering with the translation process. It specifically inhibits peptidyl transfer on both prokaryotic and eukaryotic ribosomes. The antibiotic inhibits the growth of Gram positive bacteria and various animal and insect cells. Fungi and Gram negative bacteria are resistant to Puromycin due to its low permeability. But in some particular conditions puromycin can be used for E. coli. For more than 30 years, puromycin has been widely used as a basic tool for studying protein synthesis. Now, puromycin hydrochloride is particularly useful for the selection of cell types harbouring plasmids carrying puromycin resistance genes. |
RESISTANCE TO PUROMYCIN
The pac gene encoding a Puromycin N-acetyl-transferase (PAC) has been isolated from a Streptomyces productin strain1,2. It is located in a region of the pur cluster linked to the other genes determining the puromycin biosynthetic pathway. The expression of pac gene confers puromycin resistance to transfected mammalian cells3 expressing it. Under some conditions puromycin could also be used for selection of E. coli strains transformed with plasmids carrying the pac gene.
CONDITIONS OF SELECTION
Puromycin is poorly active on E. coli but is particularly useful in experiments conducted with mammalian cells. It can be used as an alternative to the Neomycin system for transfection experiments.
The working concentrations of puromycin for mammalian cell lines range from 1 to 10µg/mL. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line. Puromycin quickly kills eukaryotic cells that do not contain the pac gene. Dying cells detach form the plates allowing easy and early identification of transformant clones.
1) VARA J., et al. (1985). Biochemistry. 24: 8074-8081
2) LACALLE R.A., et al. (1989). Gene. 79: 375-380
3) DE LA LUNA S and J. ORTIN. (1992). Methods In Enzymology. 216: 376-385
|Chemical Properties||CAS n°: 58-58.2|
Formula: C22H29N7O5, 2HCl
Molecular weight: 544.3
pKa values: 6.8 and 7.2
Puromycin is usually used at concentrations ranging from 1 to 10 µg/mL. After transformation with a plasmid containing the pac gene, cells are incubated in their regular growth medium containing Puromycin to select for stable transfectants.
1) 48 hours post-transfection, pass cells (direct or diluted) in fresh medium containing Puromycin at the appropriate concentration.
Note: Antibiotics work best when cells are actively dividing. If the cells become too dense, the antibiotic efficiency will decrease. It is best to split cells such that they are not more than 25% confluent.
2) Remove and replace antibiotic containing medium ever 3-4 days.
3) Evaluate cells for the formation of foci after 7 days of selection. Foci may require an additional week or more to develop depending on the host cell line and transfection/selection efficiency.
4) Transfer and pool 5-10 resistant clones to a 35mm cell culture plate and maintain on selection medium for an additional 7 days. This pooled culture will be expanded for subsequent cytotoxicity assays.
|Storage Condition||Shipped at room temperature. Upon receipt it should be stored at 4°C. Puromycin is stable for up to three months at room temperature and at least one year at 4°C. For optimal stability and long term storage aqueous solutions can be stored at -20°C.|
|Unit quantity||1.0 ml; 10 mg/ml|
|Will puromycin still be active in cell culture medium that is re-used for 2 to 3 weeks if stored at 4C?|
Puromycin is stable at 4C but if you add puromycin into your cell culture media and do multiple "warmings" (i.e. constantly pre-warming your media), then the puromycin in the media will degrade. Puromycin is not stable at 37C. Suggestions: 1) If you need to add the puromycin to your media, leave the media at room temperature to warm up from 4C storage instead of using the waterbath. This will keep it stable for ~ 3 weeks. 2) If water bath is needed, you can increase the puromycin concentration slightly in the media to account for overtime degradation. 3) Lastly the best way is to dilute the puromycin freshly and then add to the cells in media to the correct concentration.