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.
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.
Specificity: This antibody recognizes puromycin.
Tested Applications: Western blotting (1:1,000), ELISA and Immunofluorescence microscopy.
Storage: +4C (short-term), -20C (long-term); Avoid repeated freeze/thaw cycles.
Applications: Western blotting (1:1,000), ELISA and Immunofluorescence microscopy.
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. See protocols above for additional details.
Data: (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.
Kelleher AR, Kimball, SR, Dennis MD, Schilder RJ and Jefferson LS. The mTORC1 signaling repressors REDD1/2 are rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb. Am J Physiol Endocrinol Metab. 304(2):E229-236. 2013.
Yasuda K, Zhang H, Loiselle D, Haystead T, Macara IG, Mili S. The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules. J Cell Biol. 2013 Dec 9;203(5):737-46.
Dai N, Christiansen J, Nielsen FC, Avruch J. mTOR complex 2 phosphorylates IMP1 cotranslationally to promote IGF2 production and the proliferation of mouse embryonic fibroblasts. Genes Dev. 2013 Feb 1;27(3):301-12.
Belozerov VE, Ratkovic S, McNeill H, Hilliker AJ, McDermott JC. In vivo interaction proteomics reveal a novel p38MAPK/Rack1 pathway regulating proteostasis in Drosophila muscle. Mol Cell Biol. 2013 Nov 25.
Ueki N, Lee S, Sampson NS, Hayman MJ. Selective cancer targeting with prodrugs activated by histone deacetylases and a tumour-associated protease. Nat Commun. 2013;4:2735.
Ha SW, Ju D, Xie Y. Nuclear import factor Srp1 and its associated protein Sts1 couple ribosome-bound nascent polypeptides to proteasomes for cotranslational degradation. J Biol Chem. 2013 Dec 12.
Naydenov NG, Baranwal S, Khan S, Feygin A, Gupta P, Ivanov AI. Novel mechanism of cytokine-induced disruption of epithelial barriers Janus kinase and protein kinase D-dependent downregulation of junction protein expression. Tissue Barriers 1:4, e25231.
(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.
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.