Protein Protocols Protein Protocols

Radiolabeled Proteins 235
Larger pieces of gel can be used if one is comparing relative amounts of label, such as in
a pulse chase or other timed incorporation, but longer times and more metaperiodate may
be required to dissolve the gel. Recovery of label from the gel is in the 80–90% range for
proteins from 10–100 kDa (3). In most cases, the label will remain with the protein, but in
the case of periodate-sensitive carbohydrates on glycoproteins, it may be released from
the protein. This, however, does not prevent the quantification. It just does not allow the
solubilized labeled protein to be recovered for other manipulations.
4. It is necessary to cool the vials in the counting chamber before counting in order to eliminate
occasional chemiluminescence. The cause of this phenomenon was not explored, but
one should determine whether this occurs when using other scintillants or counters.
5. Figure 1 shows the results of a typical experiment using 35 S-methionine to label proteins
metabolically from the cellular slime mold, Dictyostelium purpureum, and quantify them
using the method described or by autoradiography. As can be seen, the resolution of the
method is comparable to that of the autoradiogram (see ref. 3 for similar results using
tritium). The time to process the lane by the method described here was approx 8 h,
whereas, the results of the autoradiogram took more than 3 d to obtain. Examination of the
stained gel suggests that, if one were interested in a particular protein, it would be fairly
easy to isolate the slice of gel containing that protein for quantification. This makes this
method extremely useful for comparing incorporation into a single protein over time as in
pulse/chase experiments (3).
6. The author has found that as little as 400 dpm of tritium associated with a protein could be
detected (3), which makes this method particularly useful for scarce proteins or small
samples.
References
1. Morrissey, J. H. (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure
with enhanced uniform sensitivity. Analyt. Biochem. 117, 307–310.
2. Van Keuren, M. L. Goldman, D., and Merril, C. R. (1981) Detection of radioactively
labeled proteins is quenched by silver staining methods: quenching is minimal for 14C and
partially reversible for 3H with a photochemical stain. Analyt. Biochem. 116, 248–255.
3. Springer, W. R. (1991) A method for quantifying radioactivity associated with protein in
silver-stained polyacrylamide gels. Analyt. Biochem. 195, 172–176.
4. Laemmli, U. K. (1970) Cleavage of structural proteins during assembly of the head of
bacteriophage T4. Nature 227, 680–685.