SHORT COMMUNICQTION MEASUREMENT OF PROTEIN MOBILITY IN SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), with the Laemmli discontinuous buffer system1 is widely used for molecular weight (m.wt) estimation and separation of proteins. The determination of m.wts. and identification of spedic proteins often relies on accurate measurement of the mobilities of proteins. When the mobilities of proteins in cell and tissue lysates are being determined, it is not possible to ensure that all the proteins are present in approximately the same concentration that permits optimal resolation within the loading capacity of the gel. In seeking to identify minor components in a mixture, the more prevalent proteins may I


Introduction
Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), with the Laemmli discontinuous buffer system1 is widely used for molecular weight (m.wt) estimation and separation of proteins. The determination of m.wts. and identification of spedic proteins often relies on accurate measurement of the mobilities of proteins.
When the mobilities of proteins in cell and tissue lysates are being determined, it is not possible to ensure that all the proteins are present in approximately the same concentration that permits optimal resolation within the loading capacity of the gel. In seeking to identify minor components in a mixture, the more prevalent proteins may I be loaded in amounts greater by one or even two orders of magnitude. Consequently, the question arises as to whether the leading edge, middle or trailing edge of a broad protein band detected by protein staining gives a more accurate estimation of its real mobility.

Materials and Methods
High m.wt marker proteins (myosin, i3-galactosidase, phosphoryiase b, bovine serum albumin, ovalbumin and carbonic anhydrase), low m.wt marker (bovine serum albumin, ovalbumin, glyceraldehyde 3-phosphate dehydrogenase, wxbonic anhydrase, trypsinogen, trypsin inhibitor and lactalbumin) and human immunoglobulin (IgG) were obtained from Sigma, St. Louis, Mo, USA. The marker roteins were dissolved 4 in reducing Laernmli bu+Yerl at a concentration of 0.5 mg d . IgG was dissolved in the buffer at 4 mg d l . Serial dilutions of the proteins in Laemmli buffer were made and equal aliquots wefully loaded into wells of the same capacity in a 4% Laemmli stacking gel and resolved by SDS-PAGE using a 10% separating gel. Polyacrylamide gel slabs were prepared as described by Laemmli.' Gels were prepared from a stock buffer contained 0.0625M Tris-HCl (pH 6 4 , 2% SDS, 10% glycerol, 5% of 2-mercaptoethanol and 0.001% bromophenol blue as a dye. Stacking was performed at 30V and separation at 120V in a Hoeffer slab gel electrophoresis apparatus. The gels were stained in 0.5% Coomassie blue, destained in 10% acetic acid with 45% methanol, dried and photographed. A general upward skewing of bands towards the sides of the gel, due to differences in current flowing between the middle of the gel and the sides, is sometimes seen in electrophoresis eg. Lanes 1,2 and 3 in Figure 1. However, such effects are not relevant to the main observation reported here since the migration of all bands, including the dye front, are similarly affected at the sides of the gel.

Discussion
When protein mobilities are measured after PAGE authors do not clearly state which part of a broad protein band is being used.' This is because it is an accepted practice to measure the centre of the band for calculating m.wts. The results presented here show that the trailing edge of stained bands of nine different proteins viz. myosin, /?-galactosidase, phosphorylase b, bovine serum albumin, glyceraldehyde 3-phosphate dehydrogenase, ovalbumin, carbonic anhydrase, trypsinogen and heavy chains of IgG are best for estimating mobilities and hence m.wts. Significant errors, as illustrated in the case of the IgG heavy chain, can be introduced by using either the middle or the leading edge of overloaded proteins bands for this purpose. The reasons for the observed phenomenon may be complex and are probably related to overloading the sieving capacity of the gel. The interpretation of the mobilities of siu$arly overloaded protein from autoradiography and enzymatically stained Western blots will depend on the degree of band broadening caused by the radiation and diffusion of coloured  Figure 2: Variation in the mobil;:y of the heavy chain of human IgG with protein concentration Lanes 1-8 were loaded with 375 ng, 750ng. 1.5pg, 3.1 pg, 6.25pg, 12.5pg, 2Spg, sntq 50pg of IgG respectively. The migration of the IgG heavy chain is indicated by a n arrow.
products of enzymatic reactions. However in both these procedures neither: the middle or the leading edge of the detected bands wiU yield the best estimate of mobility where proteins are overloaded.