Nowadays, famous companies like Merck and Thermo provide different ladders for native-PAGE analysis. However, basic biochemistry tells us that the movement of proteins on the native-PAGE is not only based on the molecular weight but also the PI and shape of proteins, which make them debatable products. Since many research article, even in leading journals like nature, use these products, which encourage others, it is important to discuss if the current native-PAGE ladders are reliable or misleading.
They are roughly approximate but, as you mentioned, they do not take into account the vast diversity of protein hydrodynamic radii for a given molecular weight. As you increase the hydrodynamic radius (at constant pI/MW) the amount of drag also increases. I have seen people describe this drag and solve for a Stoke's coefficient, in order to estimate migration time or distance, but this also assumes that the shape of the protein is a perfect sphere. I think what you are witnessing is that these researchers are not inclined to perform complex physics calculations, especially when things such as hydration shells also contribute to drag in incalculable ways. My guess is that they believe that any discrepancy would not have such a significant impact as to require mathematical modeling. I think in the vast majority of cases this is true, I am also quite certain that crucially important things are overlooked on a regular basis because taking them into account would too severely complicate research (ex: the entire field of glycobiology). In all honesty though, I have never seen a biologist doing physics and fluid mechanics calculations and I doubt I ever will.
Under native conditions, separation of proteins depends on many factors including size, shape, and native charge. Molecular weight markers will only show molecular weights, whereas proteins may oligomerize or have different shapes that would change their migration in a native PAGE environment. In such cases, the ladder isn't incredibly useful. You might use several internal standards/controls based upon what questions you're hoping to answer with your native PAGE analysis. Some good standard proteins are albumin from bovine serum (or bovine serum albumin), egg albumin, with well defined known tertiary and/or quaternary structures.
Native PAGE is one of the most powerful techniques for studying the composition and structure of native proteins, since both the conformation and biological activity of proteins remain intact during this technique. In Native PAGE proteins are prepared in a non-reducing and non-denaturing sample buffer (neither SDS nor BME in the sample buffer and in the gel), which maintains both the proteins’ secondary structure and native charge density, so this technique can could be used, for example, to know the aggregation state of a protein/ activity. Because no denaturants are used in native PAGE, subunit interactions within a multimeric protein are generally retained and information can be gained about the quaternary structure. In addition, some proteins retain their enzymatic activity (function) following separation by native PAGE. Thus, this technique may be used for preparation of purified, active proteins.
If we really want to know the molecular weight of our protein, then we need to run SDS-PAGE. In this technique, we will have both information about the number of peptides that constitute our protein and also the molecular weight.
BN page (blue native) is a form of native elettroforesis where proteins or protein complexes similar to sds page are separated on the basis of their mw and in this case the mw markers are more reilable.
Of course, native ladders can be used with a certain stretch, as they are even SDS-PAGE ladders used taking into account the possibility of the presence of isoforms that do not correspond to the intended molecular weight.
Thank you all !
In my view, at least we need different sorts of native-ladders with different range of PI in a way that people can choose the one closest to the PI of their target protein. Still, I do not know how much the shape can affect the migration and if there is a drastic difference between linear or globular proteins with the same size and PI in the movement through a native porous gel!
How do you think the native-ladders can be improved?
Hi Amir Tayaranian Marvian . I think that native PAGE ladders will be of limited general use because of differences in protein size/shape/pI, PAGE heterogeneity, PAGE crosslinking, running buffer hereogeneity, sample loading, etc.
A more practical method would be size exclusion chromatography (e.g. gel filtration).
- Badges
- Science method