Puromycin Dihydrochloride _ 60210ES
Puromycin is an aminoglycoside antibiotic produced by fermentation and metabolism of Streptomyces alboniger, which kills Gram-positive bacteria, various animal and insect cells by inhibiting protein synthesis. Puromycin is effective against E. coli in some cases. The mechanism is that puromycin is an analogue of the 3´ terminal of aminoyl-TrNA molecules, which can bind to the A site of the ribosome and be incorporated into the extended peptide chain. After puromycin binds to the A site, it will not participate in any subsequent reactions, resulting in premature termination of protein synthesis and release of immature polypeptides containing puromycin at the C-terminus. The pac gene found in the puromycin-producing strain Streptomyces alboniger encodes a puromycin N-acetyltransferase (PAC) that confers resistance to puromycin. This property is now commonly used to screen mammalian stably transfected cell lines carrying pac gene plasmids. The general application of puromycin in the selection of stable cell transfections is related to the characteristics of lentiviral vectors. Most of the commercial lentiviral vectors now carry the pac gene. In some specific cases, puromycin can also be used to screen for the transformation of E. coli strains carrying the pac gene plasmid. This product is suitable for cell culture, common working concentration is 1-10 µg/mL. In addition, Yeasen company also provides puromycin (cat# 60209ES) in solution form, which is in a different state but has the same function. Features Purity≥98% Applications Suitable for cell culture Screen specific mammalian stably transfected cell strains carrying plasmids carrying the pac gene Screen the transformed E. coli strains carrying the pac gene plasmid Specifications CAS NO. 58-58-2 Molecular Fomular C22H29N7O5·2HCl Molecular Weight 544.43 g/mol Purity ≥98% Appearance White or light white powder Structure Components Components No. Name 60210ES25 60210ES60 60210ES72 60210ES76 60210ES80 60210 Puromycin Dihydrochloride 25 mg 100 mg 250 mg 500 mg 1 g Shipping and Storage The puromycin dihydrochloride products should be stored at -15℃ ~ -25℃ for 2 years. Instructions 1. Suggested working concentration Mammalian cells: 1-10 μg/mL, optimum concentration needs to be determined by killing curve. Escherichia coli: LB agar medium was used to screen Escherichia coli stably transformed with pac gene at a concentration of 125 μg/mL. Note: Screening of stable E. coli strains using puromycin requires precise pH adjustment. Recommended concentrations of puromycin hydrochloride Cell line Concentration References B16 1-2 μg/mL [1],[2] HEK293 0.5-10 μg/mL [3] HeLa 1-10 μg/mL [4],[5] MEF 1-5 μg/mL [4] HepG2 0.5-5 μg/mL [6],[7] A549 1.5 μg/mL [8] Human embryonic stem cell(HESCS) 0.5-5 μg/mL [9] 2. Dissolution method Dissolve puromycin in distilled water to prepare a stock solution of 50 mg/mL. After sterilization by filtration through a 0.22 μm filter membrane, aliquot and store at -25 to -15℃. Alternatively, it can be dissolved in methanol to prepare a storage solution of 10 mg/mL. 3. Determination of puromycin killing curve (taking shRNA transfection or lentivirus transduction as an example) The effective screening concentration of puromycin is related to cell type, growth state, cell density, cell metabolism and cell cycle position. To screen for stably expressing shRNA cell lines, it is critical to determine the minimum concentration of puromycin that kills untransfected/transduced cells. It is recommended that customers who are doing experiments for the first time must establish a kill curve suitable for their own experimental system. 1) Day 1: The 24-well plate is plated at a density of 5-8×104 cells/well, and a sufficient number of wells are plated for subsequent gradient experiments. Cells were incubated overnight at 37℃. 2) Day 2: A) Prepare screening medium: fresh medium containing different concentrations of puromycin (such as 0-15 μg/mL, at least 5 gradients); B) Replace the freshly prepared screening medium in the cells after overnight incubation; Then the cells are incubated at 37℃. 3) Day 4: Replace with fresh selection medium and observe cell viability. 4) Depending on the growth state of the cells, change to fresh selection medium every 2-3 days. 5) Cells were monitored daily to observe the rate of viable cells to determine the lowest concentration of drug effective to kill non-transfected or all non-transduced cells within 4-6 days of the start of antibiotic screening. 4. Screening of Mammalian Stably Transfected Cell Lines After transfection of the plasmid containing the pac gene, the cells were propagated in a medium containing puromycin to select stable transfectants. 1) 48 h after transfection, the cells (original or diluted) are cultured in fresh medium containing an appropriate concentration of puromycin. Note:Antibiotics are most effective when cells are actively dividing. If the cells are too dense, the effectiveness of antibiotics will be significantly reduced. It is best to plate cells to a density of no more than 25%. 2) Remove and replace the culture medium containing puromycin every 2-3 days. 3) Cell-formed foci are assessed 7 days after screening. Lesions may require an additional week or more, depending on the host cell line and transfection screening efficiency. Note:Observe the cell growth status every day. Screening of puromycin requires at least 48 h, and the screening period of effective concentration of puromycin is generally 3-10 days. 4) Transfer and place 5-10 resistant clones into a 35 mm Petri dish and continue to culture with selection medium for 7 days. This enrichment culture is to prepare for future cytotoxicity experiment. Documents: Safety Data Sheet 60210_MSDS_HB220421_EN.pdf Manuals 60210_Manual_HB250114_EN.pdf Citations & References: [1] Furge KA. et al., 2001. Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase. PNAS 98:10722-7. [2] Díaz J. et al., 2014.Rab5 is required in metastatic cancer cells for Caveolin-1-enhanced Rac1 activation, migration and invasion.. J Cell Sci. 127:2401-6. [3] Rössger K. et al., 2013. Reward-based hypertension control by a synthetic brain-dopamine interface. PNAS, 110:18150-5. [4] Kamer I. et al., 2005. Proapoptotic BID Is an ATM effector in the DNA-damage response Cell. 122:593-603. [5] Charnaux N. et al., 2005. RANTES (CCL5) induces a CCR5-dependent accelerated shedding of syndecan-1 (CD138) and syndecan-4 from HeLa cells and forms. [6] Gao J, Zhao N, Knutson M D, et al. The Hereditary Hemochromatosis Protein, HFE, Inhibits Iron Uptake via Down-regulation of Zip14 in HepG2 Cells[J]. Journal of Biological Chemistry, 2008, 283(31):21462-8. [7] Huang J, Dibble C C, Matsuzaki M, et al. The TSC1-TSC2 complex is required for proper activation of mTOR2[J]. Molecular and Cellular Biology, 2008, 28(12):4104-4115. [8] Nasser M W, Datta J, Nuovo G, et al. Nasser MW, Datta J, Nuovo G, Kutay H, Motiwala T, Majumder S, Wang B, Suster S, Jacob ST, Ghoshal KDown-regulation of micro-RNA-1 (miR-1) in lung cancer. Suppression of tumorigenic property of lung cancer cells and their sensitization to doxorubicin-induced apoptosis by miR-1. J Biol Chem 283: 33394-33405[J]. Journal of Biological Chemistry, 2008, 283(48):33394-33405. [9] Paatero A O, Hilkka T, Happonen L J, et al. Bacteriophage Mu integration in yeast and mammalian genomes[J]. Nucleic Acids Research, 2008, 36(22): e148-e148. [10] Zhang D, et al. A non-canonical cGAS-STING-PERK pathway facilitates the translational program critical for senescence and organ fibrosis. Nat Cell Biol. 2022 May;24(5):766-782. doi: 10.1038/s41556-022-00894-z. Epub 2022 May 2. PMID: 35501370. [11] Lu T, et al. CD73 in small extracellular vesicles derived from HNSCC defines tumour-associated immunosuppression mediated by macrophages in the microenvironment. J Extracell Vesicles. 2022 May;11(5): e12218. doi: 10.1002/jev2.12218. PMID: 35524455; PMCID: PMC9077142. [12] Chen S, et al. Identification of ubiquitin-specific protease 32 as an oncogene in glioblastoma and the underlying mechanisms. Sci Rep. 2022 Apr 19;12(1):6445. doi: 10.1038/s41598-022-09497-y. PMID: 35440702; PMCID: PMC9018837. [13] Han L, et al. Uterus globulin associated protein 1 (UGRP1) binds podoplanin (PDPN) to promote a novel inflammation pathway during Streptococcus pneumoniae infection. Clin Transl Med. 2022 Jun;12(6): e850. doi: 10.1002/ctm2.850. PMID: 35652821; PMCID: PMC9161880. [14] Sun Q, et al. MORTALIN-Ca2+ axis drives innate rituximab resistance in diffuse large B-cell lymphoma. Cancer Lett. 2022 Jul 1; 537:215678. doi: 10.1016/j.canlet.2022.215678. Epub 2022 Apr 18. PMID: 35447282.
