This monoclonal antibody to puromycin provides a non-radioactive method to measure rates of global protein synthesis (mRNA translation) in cells or tissue slices incubated with puromycin, or animals treated with puromycin in vivo.
Recombinant versions available from our sister company, Absolute Antibody:
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.
From the laboratory of Scot R. Kimball, PhD, Penn State College of Medicine.
Read related blog post, Puromycin Incorporation as a Measure of Global Protein Synthesis »
EQ0001, 5: IgG1-k
Recombinant versions: see product name
|Specificity:||This antibody recognizes puromycin.|
|Purification Method:||Protein G purified|
EQ0001, 5: PBS
Recombinant versions: PBS with 0.02% Proclin 300
|Tested Applications:||Western blotting (1:1,000), ELISA and Immunofluorescence microscopy.|
|Storage:||+4C (short-term), -20C (long-term); Avoid repeated freeze/thaw cycles.|
(A) C2C12 myoblasts were starved of serum and leucine for 2 hr and then IGF-1 and leucine were added to the medium of some of the cells for 45 min. Puromycin (1uM) was added to the medium of some of the cells (lanes 3-6) 30 min before harvest. (B) Quantification of western blot analysis from panel A. (C) In the same study, but in a separate set of culture dishes, cells were incubated with [35S]methionine instead of puromycin and incorporation was measured.
A Western blot was run using the same samples where one set was run on the left side of the gel and the other on the right. The left side was probed with our original monoclonal anti-puromycin antibody and the other side was probed with the recombinant anti-puromycin antibody, both at 1:1,000 dilution. The secondary antibody was used at the same dilution for both sides and they were both exposed for ~40 sec. The first two samples were from skeletal muscle of mice where the first lane is muscle from the control hindlimb and the other is from a hindlimb that had been immobilized with a cast for three days. The other 3 lanes are from HEK393T cells: the first lane is from cells incubated in complete medium, the middle lane is from cells incubated for 2 hr in medium lacking glucose and serum, and the last lane is cells incubated for 1.5 hr without glucose or serum and then glucose and serum were returned during the last 30 min. As expected, puromycin incorporation was lower in the immobilized hindlimb compared to the contralateral control hindlimb, and also in cells deprived of glucose and serum compared to cells in complete medium. Resupplementation partially restored incorporation.
Scot R. Kimball, PhD
Penn State College of Medicine
Puromycin inhibits protein synthesis, which some cells may be more or less sensitive to. It is therefore highly recommended that the concentration of puromycin for protein translation assays be optimized for cell type. The suggested concentration of puromycin is 1uM, but for cells that are more sensitive, lower concentrations of puromycin may give better results.
Because cell growth (and protein synthesis) slows dramatically as cell near confluence, it is recommended that experiments are performed during growth phase (40-50% confluence). Label with an optimized concentration of puromycin for at least 20-30 min.
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