2D Electrophoresis With Autoradiography

Labeling proteins with radioisotopes can be very useful for studying new protein synthesis, for elucidation of turnover rates of individual proteins, and for identifying proteins with post-translational modifications such as phosphorylation. The protein mixture is resolved on a 2D gel with optional Coomassie staining and exposed
to x-ray film. Protein spots of interest can be found by matching the film to the gel and identified by mass spectrometry after the isotope has decayed to background, or immediately from a duplicate Coomassie gel. We are licensed to use ¹⁴C, ³⁵S, ³²P and ³H and will fax a copy of our license on request.

Autoradiography: Whenever possible, direct autoradiography is preferable to fluorography, especially for quantification. For purified proteins and immunoprecipitates, loads of 2500 dpm for ³²P and 5000 dpm for ¹⁴C or ³⁵S are recommended. For complex patterns, loads of 0.8-1.0 mCi ³⁵S or ¹⁴C, and 200,000 dpm ³²P are recommended for a 2 day film exposure at room temperature. ³H, a weak isotope, usually requires fluorography.

Fluorography is a method in which radioactive slab gels are permeated with a fluor prior to exposure to x-ray film at -70°C. This reduces film exposure time for ³⁵S and ¹⁴C to one fifth of that required for direct autoradiography. We recommend “ENHANCE” from New England Nuclear as the fluorographic agent. Fluorography is often used for ³⁵S- and ¹⁴C-labeled samples and almost always for ³H. The major disadvantage is that the fluor causes a faint mottling throughout the 2D pattern. Coomassie blue or silver staining prior to fluorography causes some quenching but enables the pattern to be matched to a duplicate Coomassie 2D gel for mass spectrometry.

We suggest a load of at least 500 dpm for purified or semi-purified proteins. Quite often proteins appear as a set of charge isomers with the counts spread over a wide area. When in doubt load several thousand counts to visualize minor components. For protein mixtures such as cultured cells labeled with ¹⁴C or ³⁵S, load 350,000 dpm for 2–4 day film exposures. Specific activities greater than 30,000 dpm/mg are recommended. Load 1 mCi ³H for a 2 day fluorographic exposure.

PRICE RP-1: AUTORADIOGRAPHY. Includes 2 film exposures at room temperature using 8" x 10" Kodak BioMax MR film. Exposure times are usually 2 and 4 days.  Price: Add $55 per either SF or LF 2D gel.

RP-2: ENHANCE TREATMENT. For fluorography of low energy emitters such as 3H, 14C, and 35S. (New England Nuclear reagent and protocol). The gel is soaked in a solution (Enhance) containing a fluorescent intermediate which, at low temperatures, converts beta-particle energy into photons. This treatment reduces exposure times for 14C and 35S to about 1/8 that of untreated gels. Note that disposal costs for used Enhance add to the cost.   Includes 2 film exposures at room temperature using 8" x 10" Kodak BioMax MR film. Exposure times are usually 2 and 4 days. Price: RP-2 Add $45 per SF 2D gel; RP-2LF,  Add $65 per LF gels.

RP-3: FLUOROGRAPHY. Requires prior Enhance treatment (RP-2). Includes 2 film exposures at -70oC using gel 8" x 10" Kodak BioMax MR film. Exposure times are usually 2 and 4 days. Price: Add $55 per gel.