Protein Protocols Protein Protocols

232 Springer
Fig. 1. Comparison of an autoradiogram, densitometry scan, and radioactivity in gel slices
from a mixture of labeled proteins separated by PAGE. Identical aliquots of 35 S-methioninelabeled
proteins from the membranes of the cellular slime mold, D. purpureum, were separated
using the methods described in Subheading 3.1. One lane was fixed, soaked in Flouro-Hance
(Research Products International Corp., Mount Prospect, IL), dried, and autoradiographed (photograph).
Another lane was cut from the gel, sliced into 1-mm pieces, solubilized, and counted
using a Tracor Mark III liquid scintillation counter with automatic quench correction (TM Analytic,
Elk Grove Village, IL) as described in Subheading 3.3. The resultant disintigrations per
minute (DPM) were plotted vs the relative distance from the top of the running gel (top figure).
The autoradiogram (photograph) was scanned relative to the top of the running gel using white
light on a Transidyne RFT densitometer (bottom figure).
4. Tris II: 1.5 M Tris-HCl, pH 8.8, 0.4% SDS.
5. Tris III: 0.5 M Tris-HCl pH 6.8, 0.4% SDS.
6. Ammonium persulfate (APS) solution: A small amount of ammonium persulfate is
weighed out and water is added to make it 100 mg/mL. This should be made fresh the day
of the preparation of the gel. The ammonium persulfate crystals should be stored in the
refrigerator and warmed before opening.
7. N,N,N',N'-tetramethylethylenediamine (TEMED) is used neat as supplied by the
manufacturer.
8. 10X Running buffer: Tris base (30.2 g), glycine (144.1 g), and SDS (10.00 g) are made up
to 1 L with distilled water.