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Focusing on improved SDS-PAGE separations focusing gels were prepared in standard gradient casters by incorporation of positively charged ions, called Immobilines, into acrylamide matrices during poly0 mM pK 10.3 2 kDa
Migration
Imagine a parallel universe where the laws of physics are turned upside down—where large proteins migrate faster than small proteins on an SDSPAGE gel, and researchers can easily separate small peptides by SDS-PAGE. In our own universe, such separations have proven to be challenging, if not impossible. But now, without defying a single law of physics, Shmuel Bukshpan, Gleb Zilberstein, Pier Giorgio Righetti, and colleagues at Cleardirection Ltd. (Israel) and Politecnico di Milano (Italy) describe in this issue of Analytical Chemistry (pp 821–827) a new form of SDS-PAGE called SDS-PAGE focusing. The technique separates proteins in an unorthodox manner, with large proteins migrating faster than small ones. The new approach is a hybrid between SDS-PAGE and isoelectric focusing (IEF). In conventional SDS-PAGE, micelles of the anionic surfactant SDS bind to polypeptide chains, overwhelming intrinsic charges so that most proteins adopt a uniform net negative charge per unit of mass. Proteins are separated in a sieving matrix on the basis of molecular mass, with small proteins migrating more rapidly toward the anode than large proteins. Since the refinement of SDS-PAGE in the early 1970s, this technique has been the workhorse for determining molecular masses of proteins, for assessing protein purity, and as a second dimension in 2D protein separation. However, two major limitations of SDSPAGE are its failure to separate small peptides and its inability to resolve proteins with similar molecular masses. Bukshpan and colleagues devised their method for improved SDS-PAGE separation by adopting principles of IEF, an electrokinetic approach that uses a pH gradient gel with large pores to separate proteins on the basis of intrinsic charge. In the SDS-PAGE focusing technique, the investigators grafted a gradient of positive charges onto a dilute polyacrylamide gel. The SDS-PAGE
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18 mM pK 10.3
SDS-PAGE focusing of protein–SDS complexes in a gel containing a gradient of positive charges. Because large proteins migrate farther than small ones, this technique provides better separation of small proteins than conventional SDS-PAGE does.
merization; this imparted a positive charge to the otherwise neutral polyacrylamide matrix. In contrast to immobilized pH gradient gels used in IEF, negatively charged counterions were not grafted onto the gel. In this set-up, SDS acted as a counterion in the form of soluble protein–SDS micelles. When the micelles reached a gel zone where charge density equaled the total negative charge on the surface of the complex, protein migration stopped. Because charge density in the SDSPAGE focusing gel increased in the cathode-to-anode direction, large polypeptides migrated farther than small polypeptides in the gel. This counterintuitive electrophoretic migration pattern was in direct contrast to conventional SDS-PAGE. For SDS-PAGE focusing, the plot of Mr versus migration distance had a positive slope and linear-scaled axes, as opposed to a negative slope and
a logarithmic M r axis for conventional SDS-PAGE plots. The linear scale allowed better accuracy in the mass determination over a large mass range. A major advantage of SDS-PAGE focusing over conventional SDS-PAGE was its enhanced ability to separate small peptides. Bukshpan says, “In 2D map analysis, all peptides in the size range
