What is the function of puromycin?
Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating nascent chains, blocking further extension and resulting in premature termination of translation.
Why is puromycin used in cell culture?
Puromycin is an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. It inhibits protein synthesis by disrupting peptide transfer on ribosomes causing premature chain termination during translation. It is a potent translational inhibitor in both prokaryotic and eukaryotic cells.
How does puromycin selection work?
This compound works by interrupting protein synthesis in both prokaryotic and eukaryotic cells. This causes cells to die, unless they have a gene for resistance, allowing them to survive exposure to the antibiotic.
How much puromycin do I add?
In mammalian cells, the recommended working concentration range for puromycin is 0.5 – 10 µg/ml. Different cell types and cell culture conditions may require different concentrations of selection antibiotic.
What does puromycin do in translation?
Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor.
How long is puromycin selection?
Puromycin selection requires a minimum of 48 hours. Optimum effectiveness should be reached within 3-10 days. Assay transfected cells.
How do I select cells with puromycin?
You want your selection with puromycin to be done within a week. So you should select a concentration of puromycin that would kill all untransfected cells in one week or less and leave only the transfected cells that will live and be stably transfected.
How long does puromycin last?
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.
How long does puromycin selection take?
What is puromycin labeling?
When should I start puromycin selection?
The selection (addition of puromycin to the cell culture media) is usually started around 24 hr following transfection. You can also start the selection at 48 hr depending on the health of your transfected cells.
What is the puromycin resistance gene?
Resistance to puromycin is conferred by the pac gene encoding a puromycin N-acetyl-transferase (PAC) that was found in a Streptomyces producer strain. Puromycin is soluble in water (50 mg/ml) as colorless solution at 10 mg/ml. Puromycin is stable for one year as solution when stored at -20 °C.
Is puromycin an aminoglycoside?
Puromycin dihydrochloride is the hydrochloride salt form of puromycin, an aminoglycoside antibiotic isolated from the bacterium Streptomyces alboniger.
Does puromycin affect eukaryotes?
Puromycin is used in cell biology as a selective agent in cell culture systems. It is toxic to prokaryotic and eukaryotic cells.
What are the 7 main classes of antibiotics?
In this portal, antibiotics are classified into one of the following classes: penicillins, fluoroquinolones, cephalosporins, macrolides, beta-lactams with increased activity (e.g. amoxicillin-clavulanate), tetracyclines, trimethoprim-sulfamethoxazole, lincosamides (e.g. clindamycin), urinary anti-infectives, and other …
What is anti-puromycin antibody?
Anti-Puromycin antibody, clone 12D10, detects puromycin incorporated into protein. Monoclonal antibodies to puromycin may be used with standard immunochemical methods. More>> Anti-Puromycin antibody, clone 12D10, detects puromycin incorporated into protein.
What are monoclonal antibodies to puromycin?
Monoclonal antibodies to puromycin may be used with standard immunochemical methods to directly monitor translation, a method known as surface sensing of translation (SUnSET). Part of the molecule resembles the 3′ end of the aminoacylated tRNA, making it useful for protein translation analysis.
Which gene is expressed in MCF7 adhesive cell line?
Transformed from human breast cancer cell line, MCF7 adhesive cell using lentivirus expressing firefly luciferase gene. The Luciferase was expressed under the enhanced EF1a promoter. The Puromycin marker was expressed under Rsv promoter.
What is the mechanism of action of puromycin?
Abstract Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating nascent chains, blocking further extension and resulting in premature termination of translation.