Overview
Cell-based radioactive phosphorylation assays rely on the use of phosphate-free culture media supplemented with a radiolabeled source of phosphate to "feed" the cells, 32P. Typically, the radiolabeled phosphorus is supplied in the form of orthophosphate (Pi), rather than radiolabeled ATP (in general, it is more difficult to permeate cells with ATP). Identification of phosphorylation of a specific target substrate is usually carried out by immunoprecipitating the target-of-interest, then running on an SDS-PAGE gel and exposing to film (autoradiography) or phosphorimaging (though other methods of substrate extraction and detection are also possible).
What do I need to run this assay?
- 32Pi (orthophosphate)
- Cells
- Phosphate-free culture media (or other feeding/treatment buffer)
- Tissue culture equipment
- Immunoprecipitation column and reagents
- Gel electrophoresis reagents and equipment
- Film for autoradiography (unless using a phosphorimager)
- Film developer, or a phosphorimager as appropriate
Products
Radiolabeled orthophosphate and phosphate salts
Refer to the information below this table for guidance in choosing a product for your assay.
| Product | Isotope | Diluent | Specific activity | Rad. conc. |
|---|---|---|---|---|
| Disodium phosphate | 32P | Water | 900-1100 mCi/mmol | Varies depending on amount of radioactivity ordered - packaged in 1 mL water, no matter size |
| Monopotassium phosphate | 32P | Water | 900-1100 mCi/mmol | Varies depending on amount of radioactivity ordered - packaged in 1 mL water, no matter size |
| Monosodium phosphate | 32P | Water | 900-1100 mCi/mmol | Varies depending on amount of radioactivity ordered - packaged in 1 mL water, no matter size |
Guidance for using above table:
- Most protocols use orthophosphate rather than a phosphate salt for labeling
- If your protocol calls for carrier-free 32P use NEX053/NEX053C/NEX053H.
- Higher specific activity indicates that there is more radioactivity per millimole or milligram of phosphorus. Units are given either as Curies per millimole of phosphorus, or Curies per milligram of phosphorus. As you approach the theoretical maximum specific activity for a radionuclide (~9120 Ci/mmol for 32P), a greater proportion of phosphorus in the stock vial is labeled. Products at maximum theoretical specific activity for a given isotope are termed "carrier-free". Specific activity can be lowered by adding cold phosphate.
- For the products below, use the molecular weight of phosphorus itself (rather than the phosphate salt) to convert from Ci/mmol to Ci/mg, and vice versa
Autoradiography enhancers for film or phosphorimaging
- EN3HANCE™ Autoradiography Enhancer (Cat. No. 6NE9701): use on gels for best band resolution
- ENLIGHTNING™ Rapid Autoradiography Enhancer (Cat. No. 6NE9741): safer alternative to 6NE9701. Use on gels.
Detection instruments
- Phosphorimager
Protocol-in-brief
Workflow
Citations
- Cooke, F.T. Measurement of phosphoinositide 3-kinase products in cultured Mammalian cells by HPLC. Methods Mol. Biol 645, 179-202 (2010). Link
- Foltyn, V.N., Zehl, M., Dikopoltsev, E., Jensen, O.N. & Wolosker, H. Phosphorylation of mouse serine racemase regulates D-serine synthesis. FEBS Lett 584, 2937-2941 (2010). Link
- Neddermann, P. NS5A phosphorylation and hyperphosphorylation. Methods Mol. Biol 510, 95-110 (2009). Link
- Strachan, R.T. et al. Ribosomal S6 kinase 2 directly phosphorylates the 5-hydroxytryptamine 2A (5-HT2A) serotonin receptor, thereby modulating 5-HT2A signaling. J. Biol. Chem 284, 5557-5573 (2009). Link
Other Revvity cellular kinase technologies
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