Puromycin Dihydrochloride-selection antibiotic-TOKU-E

Detailed Description

Puromycin Dihydrochloride (syn: Puromycin DiHCl) is the hydrochloride salt of Puromycin, an aminonucleoside antibiotic with anti-trypanosomal and anti-neoplastic properties. Puromycin was isolated from Streptomyces alboniger in the 1950s. Puromycin DiHCl is routinely used in cell culture as a selective agent in transfection and transformation protocols to select for cells that have been transformed with the Puromycin resistance gene (pac) and express puromycin-N-acetyl-transferase.

We also offer:

Puromycin (P097)
Puromycin Aminonucleoside (P041)
Puromycin DiHCl Solution (10 mg/ml in 20 mM HEPES)(P025-P026)

Mechanism of Action	Puromycin Dihydrochloirde inhibits protein synthesis in two ways: 1) Puromycin associates with the donor substrate, peptidyl-tRNA, in the P site and functions as an acceptor substrate. 2) Puromycin DiHCl can compete with aminoacyl tRNA to bind with the A′ site within the peptidyl transferase center causing premature chain termination. Resistance to Puromycin is conferred by the pac gene, a 60 nt fragment that encodes puromycin N-acetyltransferase. The enzyme inactivates Puromycin by acetylating the amino group in the tyrosinyl moiety. Acetylated Puromycin is biologically inactive and does not associate with prokaryotic or eukaryotic ribosomes.
Spectrum	Puromycin can prevent growth of bacteria, algae, protozoa, and mammalian cells and acts quickly, killing 99% of cells within 2 days. Puromycin DiHCl is active against both prokaryotic and eurkaryotic cells. It is active against Gram-positive bacteria and less active against Gram-negative and acid-fast bacilli.
Microbiology Applications	Puromycin Dihydrochloride can be used to select for Puromycin resistant bacteria that have been transformed with the pac gene. Resistant E. coli transformants can be isolated on pH adjusted LB medium using a Puromycin concentration of 100-125 µg/mL. Puromycin DiHCl can also be used as a selectable marker in mollicute research and has been successfully used to select for various Mycoplasma species after transformation with the Puromycin resistance gene (pac). Tetracycline is traditionally used as a selectable maker for Mycoplasma; however, Puromycin does not have any clinical value, is a potent protein synthesis inhibitor, and can be used to screen for a wide range of Puromycin resistant Mycoplasma spp. Because of its unique mechanism of action, there is a low possibility of spontaneous resistance to Puromycin by a point mutation.
Eukaryotic Cell Culture Applications	Puromycin Dihydrochloride has been used to screen cells transfected cells with the CRISPR/Cas system, by co-transfecting a resistance plasmid, and screening with Puromycin as a selection marker. Effective working concentrations for selection of puromycin resistant cells range from 0.5 – 10 µg/mL. The optimal working concentration of Puromycin Dihydrochloride for selection of resistant mammalian clones depends on the cell lines used, Puromycin quality, media, growth conditions, cell density, cell metabolic rate, cell cycle phase, and the plasmid carrying the pac resistance gene. A kill curve should therefore be performed to determine the optimal working concentration for every experimental system and for every lot of Puromycin dihydrochloride. Optimal selection concentrations of Puromycin typically range from 0.5 µg/mL - 2 µg/mL for suspended cells and 2 µg/mL - 5 µg/mL for adherent cells. For Puromycin kill curve protocol, click here. For additional information regarding relevant cell lines, resistance plasmids, and culture media, please visit our cell culture database.
Cancer Applications	Puromycin Dihydrochloride has shown anti-tumor activity when tested against numerous cell lines. A novel prodrug strategy targeting increased histone deaceylase (HDAC) and endogenous protease cathepsin L (CTSL) activities in malignant tumors. The cancer-selective cleavage of the small-molecule masking group may be a strategy for anticancer compound development. By coupling an acetylated lysine group to Puromycin (from TOKU-E), a masked cytotoxic agent is created, which is activated by HDAC and CTSL that removes the acetyl group followed by the unacetylated lysine group, liberating Puromycin. This could be a promising strategy for anticancer compound development (Ueki et al, 2013). Ring finger protein 43 (RNF43) is known for its role in negative regulation of the Wnt-signaling pathway in cancer. However, the function in DNA double-strand break repairs has not been investigated. Researchers used cellular models (lymphoblast cell line, DT40 along with mouse embryonic fibroblast) to study DNA double-strand break (DSB) repairs. They used Puromycin DiHCl from TOKU-E EU for selection of transfectants for RNF43^-/- screening (Lerksuthirat et al, 2020).
Molecular Formula	C₂₂H₂₉N₇O₅ · 2HCl
References	Conti et al. used Puromycin DiHCl (TOKU-E) to select for eGFP expressing A549 cells. "Polymeric nanocarriers and their oral inhalation formulations for the regional delivery of nucleic acids to the lungs." Lerkusuthirat et al. used Puromycin DiHCl (TOKU-E) for selection of transfectants for RNF43 -/- screening in cancer research. ''A DNA repair player, ring finger protein 43, relieves etoposide-induced topoisomerase II poisoning.'' Lu et al. used Puromycin DiHCl (TOKU-E) to select for transfected AS-B145 and BT-474 cells. "Ovatodiolide inhibits breast cancer stem/progenitor cells through SMURF2-mediated downregulation of Hsp27" Sandoval-Jaime et al. used Puromycin DiHCl (TOKU-E) to select for stably transfected cells. "Recovery of murine norovirus and feline calicivirus from plasmids encoding EMCV IRES in stable cell lines expressing T7 polymerase." Algire MA (2009) New selectable marker for manipulating the simple genomes of Mycoplasma species. Antimicrob. Agents and Chemother. 53(10):4429-4432 Azzam ME (1973) Mechanism of Puromycin action: Fate of ribosomes after release of nascent protein chains from polysomes." PNAS 70.12:3866-3869. Lacalle R et al (1989) Molecular analysis of the pac gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger. Gene. 79:375-380 Lerksuthirat T et al (2020) A DNA repair player, ring finger protein 43, relieves etoposide-induced topoisomerase II poisoning. Genes Cells. 25:718–729 Ueki N et al (2013) Selective cancer targeting with prodrugs activated by histone deacetylases and a tumor-associated protease. Nat. Commun 4:2735 Vara J (1985) Cloning and expression of a Puromycin N-acetyl transferase gene from Streptomyces alboniger in Streptomyces lividans and Escherichia coli. Gene 33(2):195-206
Protocols	Puromycin Dihydrochloride Kill Curve Protocol Background: Puromycin Dihydrochloride (DiHCl) is an aminonucleoside antibiotic derived from Streptomyces alboniger and is routinely used as a selective agent for mammalian cells that have been transformed or transfected with plasmids containing the puromycin resistance gene, pac. Before stable transfected cell lines can be selected, the optimal Puromycin DiHCl concentration needs to be determined by performing a kill curve titration. The optimal concentration of Puromycin DiHCl suitable for selection of resistant mammalian clones depends on the cell lines, media, growth conditions, and the quality of Puromycin DiHCl but typically lies between 1 µg/mL - 10 µg/mL. Because of these variables, it is necessary to perform a kill curve for every new cell type and new batch of Puromycin DiHCl. Preparation and storage of Puromycin DiHCl solution: Puromycin DiHCl is soluble in water at 50 mg/mL yielding a clear, colorless to faint yellow solution. Store filter-sterilized (0.22 μm) stock solutions solutions in aliquots at –20 °C. Kill curve/Puromycin DiHCl titration protocol: Seed cells of the parental cell line in a 24-well plate at different densities (50,000 – 100,000 and 200,000 cells/ml) and incubate the cells for 24 hours at 37°C. Remove medium and then add medium with varying concentrations of antibiotic (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μg/ml) and incubate at 37°C. Refresh the selective medium every 3-4 days and observe the percentage of surviving cells over time (e.g. by EMA vs Hoechst staining, flow cytometry, or MTT assay). Determine the lowest concentration of antibiotic that kills a large majority of the cells within 14 days. This concentration should be used for selection of a stable transfected cell line. 5. If necessary, repeat the experiment to narrow the antibiotic concentration range. Click here for PDF Plasmid DNA Transfection Protocol Background: Once the appropriate antibiotic concentration to use for selection of the stable transfected cells has been determined by performing a kill curve, the next step is to generate a stable cell line by transfection of the parental cell line with a plasmid containing the gene of interest and an antibiotic resistance gene. Plasmid DNA Transfection Protocol: Seed the parental cell line in 24-well plate and incubate for 24h at 37°C. Transfect the parental cell line the next day at 80% confluency with the construct (e.g. using calcium phosphate etc…) and include a sample of untransfected cells as a negative control. Incubate at 37°C in C0₂. After transfection (6h to 24h depending on the transfection method used), wash the cells once with 1X PBS and add fresh medium containing the selection antibiotic to the cells. Use the appropriate antibiotic concentration as determined from the kill curve. Check, refresh, and expand the cells in selection medium every 2-3 days until you have enough cells for limited dilution (confluency in T25 flask or 10 cm dish). QC Seed 24-wells with insert and determine the transfection efficiency by immunostaining: Grow cells on insert in a 24-well plate until well is confluent. Remove medium and wash cells with 1X PBS. Fix cells with methanol or paraformaldehyde and wash with 1X PBS. Add primary antibody in 24-well against protein of interest and incubate at 37°C for 1 hour (depending on antibody). Wash cells with 1X PBS. Add secondary antibody in 24-well and incubate at 37°C for 1 hour (depending on antibody). Wash with 1X PBS. Remove insert from 24-well plate and affix to microscopy slide with clear nail polish or other suitable adhesive. Determine the percentage of transfected cells with fluorescence microscope. Click here for PDF Selection of Stable Transfected Cell Lines Protocol Background: Once the cells have been successfully transfected, the next step is to seed and select the transfected cell line in a single 96-well plate to select pure colonies by limited dilution as outlined below: Protocol: Seed the transfected cells in 96-well plates in 10% conditioned medium 2x96 well plate with 0.1 cell per well 2x96 well plate with 0.5 cell per well 2x96 well plate with 1 cell per well Incubate the cells for 24h. Remove medium and add conditioned selection medium containing selection antibiotic at the pre-determined concentration required for your cell line. Incubate 96-well plates at 37°C with C0₂. Check the plates every day for colonies. Colony formation depends on proliferation rate of the cell line and can take anywhere from 3 days to 1 week. Refresh selective medium every 3-4 days until colonies appear. Select the wells with only one single colony. Make sure colonies are not growing in clumps as they will be less sensitive to the antibiotic. When a well contains a single colony, transfer the colony to a 24-well in selection medium and so on until you have enough cells for freezing and storage in liquid nitrogen. Use the appropriate antibiotic concentration as determined from the kill curve. QC Seed 24-wells with insert for an immunostaining to determine percentage of cells expressing the gene of interest to be able to identify a 100% pure clone. You can also use Western blotting, flow cytometry or another technique depending on the cell line used. Seed 24-wells with insert and determine the expression level of the gene of interest by immunostaining: Grow cells on insert in a 24-well plate until well has confluent growth. Remove medium and wash cells with 1X PBS. Fix cell with methanol or paraformaldehyde and wash with 1X PBS. Add primary antibody in 24-well against protein of interest and incubate at 37°C for 1 hour (depending on antibody). Wash cells with 1X PBS. Add secondary antibody in 24-well plate and incubate at 37°C for 1 hour (time depends on antibody type). Wash cells with 1X PBS. Remove insert from 24-well plate and affix to microscopy slide with clear nail polish or other appropriate adhesive. Determine the percentage of transfected cells with fluorescence microscope. Click here for PDF.