Anomalous Migration in Sdspage

The precise structural relationship of SDS to protein during SDS-PAGE is unknown but various studies have indicated that a wide variety of proteins all bind a relatively constant amount of SDS (+1.4 g SDS g_1 protein) and adopt a similar flexible rod shape regardless of their native configuration. It is this supposedly constant very high and uniform charge-mass ratio which allows for reliable molecular weight determination since migration rate therefore depends on molecular sieving alone. Having said this, even the original paper of Laemmli referred to the anomalously slow migration of a bacteriophage point mutant. The very basic proteins, histones, behave so anomalously that special buffer conditions in the presence of urea must be employed to determine reliably their molecular weights. Glycoproteins also show anomalous migration. In this laboratory we have, over the years, seen direct evidence of discrete changes in apparent migration in SDS-PAGE related to single mutations which change the charge on a protein without significantly affecting its calculated molecular weight. Figure 5, for instance, shows an SDS-PAGE separation of purified carbonic anhydrase 1 (CA1) from a Caucasian blood donor (lane 1) or an Australian Aboriginal donor (lane 4) who is heterozygous for a polymorphic variant CA1-9. The normal and the variant component of the heterozygote were resolved by isoelectric focusing (lanes 2 and 3, respectively). The variant component differed from the normal component at only one position (Asp p Gly) although the apparent molecular weight after SDS-PAGE was 27 kDa compared with 28.5 Da. From this and other previous studies with defined point mutations of defined proteins we propose that each extra negative charge on a modified protein results in retardation on SDS-PAGE such that its apparent molecular weight is greater by 1.5 kDa.We have applied this rule of thumb to changes in the apparent molecular weights of the keratins of human hair after S-car-boxymethylation which substitutes an extra negative charge for each thiol. See Figure 1 for general molecular weight shift of hair proteins after S-car-boxymethylation - compare the profile from lane 4 (red) with the profile from lane 2 (blue). Given the published molecular weights of the unmodified keratins we were then able to calculate an apparent thiol content in rough agreement with previous estimations assuming that each thiol group was substituted with an extra negative charge after carboxymethylation (lane 2).

We are therefore convinced that electrophoretic migration of SDS-protein complexes is not totally independent of charge of the native protein and furthermore that if a one-dimensional SDS-PAGE system could be suitably calibrated, the relationship we have described could become useful in studies where the net charge on a given protein is changed genetically or epigenetically in an incremental way.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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