Puromycin is an aminonucleoside antibiotic, derived from the Streptomyces alboniger bacterium, that causes premature chain termination during translation taking place in the ribosome. Part of the molecule resembles the 3' end of the aminoacylated tRNA, making it useful for
protein translation analysis.
Classical pulse-chase or flooding dose methods used to monitor protein synthesis rely on the measurement of radioactive methionine and cysteine labels. Analysis using puromycin immunodetection is an advantageous alternative to radioactive amino acid labeling, and allows for the evaluation/quantification of translation directly using standard immunochemical methods.
|Applications||Western Blotting (1:1,000), ELISA (1:10,000) and Immunofluorescence microscopy|
Human fibroblast (TIG-1 cells) was plated to 12-well plate (Cells were cultured till they became 80% confluent).
Puromycin (final conc. 0 uM and 5 uM) was added to the medium of the cells 80 min before harvest.
Wells were washed with PBS, followed by addition of 1mL buffer (20 mM Tris-HCl(pH8.0)+Protease Inhibitor) to each wells to perform ultrasonic fragmentation.
Cells were spun down (4℃, 12,000 g, 20 min), supernatant was collected for downstreaam assays (Western Blotting, ELISA).
Loaded 15 ug/lane
Primary Antibody (anti puromycin [CAC-PEN-MA001]) 1:1,000 dilution (1 ug/mL)
Secondary Antibody (anti mouse Ig-HRP, DAKO, Cat.No. P0161)1:2,000 dilution