SKU: P025-P026  / 
    CAS Number: 58-58-2

    Puromycin Dihydrochloride ReadyMade™ Solution

    RM876.38 - RM2,344.13
    Puromycin Dihydrochloride ReadyMade™ Solution

    Puromycin Dihydrochloride ReadyMade™ solution (10 mg/mL in 20 mM HEPES) is an aminonucleoside antibiotic solution derived from Streptomyces alboniger. Puromycin DiHCl is routinely used as a selective agent in transfection and transformation protocols.

    For more Puromycin products, click here.

     

    Mechanism of Action During translation, Puromycin enters the ribosomal “A” site and disrupts peptide transfer. As a result, the ribosome stops and the peptide chain is terminated leading to a nonfunctional protein.
    Spectrum Puromycin DiHCl is active against both prokaryotic and eurkaryotic cells.
    Eukaryotic Cell Culture Applications Puromycin DiHCl is commonly used as a selective agent to isolate mammalian cells after transfection with the pac gene. The pac gene encodes puromycin N-acetyltransferase; a protein that inactivates Puromycin by acetylation.

    Conti et al. used Puromycin DiHCl solution (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.

    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.

    Mutonga et al. used Puromycin (TOKU-E) to select for resistant cells transformed with a vector containing SUV39H2 (a histone methyltransferase) and a puromycin resistance gene. Targeting suppressor of variegation 3-9 homologue 2 (SUV39H2) in acute lymphoblastic leukemia (ALL)." 

    Foltyn et al. used Puromycin, G418 Disulfate and Hygromycin B (TOKU-E): The physiological mTOR complex 1 inhibitor DDIT4 mediates therapy resistance in glioblastoma.

    For more information on relevant cell lines, media, and working concentrations, please visit the TOKU-E Cell Culture Database.
    Molecular Formula C22H29N7O5•2HCl
    Documents Puromycin Protocol.pdf|Plasmid_DNA_Transfection_Protocol.pdf|Selection_of_Stable_Transfected_Cell_Lines_Protocol.pdf
    References

    Azzam ME (1973)  Mechanism of Puromycin action: Fate of ribosomes after release of nascent protein chains from polysomes. PNAS 70(12):3866-3869

    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 DiHCl Kill Curve Protocol

    Background:

    Puromycin 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. The stock solution may be passed through a 0.22 μm filter and stored in aliquots at –20 °C.

    Kill curve/Puromycin DiHCl titration protocol:

    1. 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.
    2. 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.
    3. 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).
    4. 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:

    1. Seed the parental cell line in 24-well plate and incubate for 24h at 37°C.
    2. 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.
    3. 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.
    4. 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:

    1. Grow cells on insert in a 24-well plate until well is confluent.
    2. Remove medium and wash cells with 1X PBS.
    3. Fix cells with methanol or paraformaldehyde and wash with 1X PBS.
    4. Add primary antibody in 24-well against protein of interest and incubate at 37°C for 1 hour (depending on antibody).
    5. Wash cells with 1X PBS.
    6. Add secondary antibody in 24-well and incubate at 37°C for 1 hour depending on antibody).
    7. Wash with 1X PBS.
    8. Remove insert from 24-well plate and affix to microscopy slide with nail polish or other suitable adhesive.
    9. Determine the percentage of transfected cells with fluorescence microscope.
    Click here for PDF

    Selection of Stable Transfected Cell Lines

    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:

    1. 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
    2. Incubate the cells for 24h.
    3. 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.
    4. 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.
    5. Refresh selective medium every 3-4 days until colonies appear.
    6. 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.
    7. 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:

    1. Grow cells on insert in a 24-well plate until well has confluent growth.
    2. Remove medium and wash cells with 1X PBS.
    3. Fix cell with methanol or paraformaldehyde and wash with 1X PBS.
    4. Add primary antibody in 24-well against protein of interest and incubate at 37°C for 1 hour (depending on antibody).
    5. Wash cells with 1X PBS.
    6. Add secondary antibody in 24-well plate and incubate at 37°C for 1 hour (time depends on antibody type).
    7. Wash cells with 1X PBS.
    8. Remove insert from 24-well plate and affix to microscopy slide with nail polish or other appropriate adhesive.
    9. Determine the percentage of transfected cells with fluorescence microscope.

    Click here for PDF.
    MIC Streptococcus pneumonia (ATCC 6303 + quinolone-susceptible)| 1 － ?| 437| Streptococcus pneumonia (ATCC 7257 + quinolone-resistant)| 1 － ?| 437|