The solubilization buffer most commonly used to prepare samples for SDS polyacrylamide gel electrophoresis is that described by Laemmli (Nature 227:680-685). While this solubilization buffer works well for the electrophoretic analysis of most proteins, we find that solubilization of thylakoid membrane polypeptides in Laemmli's buffer leads to a time and temperature-dependent loss of a spectrum of lamellar polypeptides. These losses are clearly seen in Figure 1A where aliquots from a single thylakoid preparation were solubilized in Laemmli's buffer at 4 C, 20 C, or 100 C for 10 minutes and subjected to PAGE at 4 C. The loss of polypeptides A-E at 100 C is accompanied by the appearance of high molecular weight material at the top of the analyzing gel, suggesting that these polypeptides aggregate when heated in the sample buffer. These losses occur at progressively lower temperatures as thylakoids are incubated in the solubilization buffer for longer periods of time (compare polypeptide A in Figs. 1A, 1B). Raising the run temperature from 2 C to 8 C or increasing the current to induce gel heating also promotes the loss of polypeptides A-E. The polypeptides lost after heating represent core components of both photosystems I and 11. Note that polypeptides F and G increase in intensity after heating; this is consistent with the known action of LDS on multisubunit and chlorophyll-protein complexes.
We find that the loss of polypeptides A-E is minimized by replacing the buffer species (tris, pH 6.8) and sulfhydryl reagent (2-mercaptoethanol) in Laemmli's formulation with tricine, pH 7.8, and dithiothreitol, respectively, as suggested by Jim Metz (Biochim. Biophys. Acta 681:95-102). With these changes, the improved retention of polypeptides A-E is evident (Figs. 1C, 10). We have varied the pH (6.8 vs. 7.8), reductant (2-mercaptoethanol vs. dithiothreitol), buffer (tris vs. tricine) and detergent (SDS vs. LDS) separately and in combination and find that the pH of solubilization has the greatest effect on stability. We now use the sample buffer described in Figure 1C for routine analysis of thylakoid membrane polypeptides. Lithium dodecyl sulfate is preferred over the sodium salt in this system because of its greater solubility at low temperature. Sample solubilization is carried out at 4 C for 10 minutes or by heating to 100 C for 2 minutes, followed by electrophoresis at 2-4 C in a jacketed apparatus (Bio-Rad).
All samples were solubilized at detergent: chl ratio of 20:1 (w/w). Arrows indicate polypeptides appearing or disappearing as a function of solubilization conditions.
Kenneth J. Leto and Roslyn Young
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