Anti Puromycin Monoclonal Antibody (3RH11)
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. The puromycin is detected by either Western blot or ELISA.
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 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.
See below for suggested use.
From the laboratory of Scot R. Kimball, PhD, Penn State College of Medicine
Part of The Investigator's Annexe program.
| Product: ||Anti-Puromycin (3RH11) |
| Clonality: ||Monoclonal |
| Specificity: ||This antibody recognizes puromycin. |
| Immunogen: ||puromycin hydrochloride |
| Raised in: ||Mouse |
| Isotype: ||IgG1 kappa |
| Purity: ||Protein G purified |
| Form: ||Liquid |
|Concentration: ||1 mg/ml |
| Volume: ||100uL |
| Applications: ||Western blotting (1:1,000), ELISA and Immunofluorescence microscopy. |
| Shipped: ||Shipped on cold pack |
|Storage conditions: ||Store at 4C |
Protocol for Measuring Protein Synthesis with Puromycin via Western Blot
Protocol for Measuring Protein Synthesis with Puromycin via ELISA
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 experimetns 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.
- Hoeffer, CA et al. Inhibition of the interactions between eukaryotic initiation factors 4E and 4G impairs long-term associative memory consolidation but not reconsolidation. PNAS. 108(8): 3383 - 3388. 2011.
- Goodman, CA et al. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J. 25: 1028 - 1039. 2011.
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