Size and Shape

The sizes of protein molecules vary over a large range, but most have a molecular weight 20000-200000 Da (native protein). This translates to a size, assuming a perfect sphere, of roughly 3-8 nm in diameter. Many proteins are not spherical, however, so their longer dimensions will be greater than these figures. Separation by size is carried out by a process called gel filtration or size exclusion chromatography. The principle of gel filtration is shown in Figure 2. Beads that are porous, with pores of similar size to the proteins, are packed into a column. The largest proteins cannot penetrate the beads because the pores are too small, so they flow quickly around the outside of the beads, and emerge first.

They do not emerge until after a volume (the void volume, V0) that is equivalent to the volume of liquid outside the beads has passed through the column. This volume is typically about 30% of the total column volume, Vt. The smallest proteins are able to penetrate the pores in the beads. While in the beads they are isolated from the flow of liquid (which cannot pass through the beads because of capillary forces), and so do not move down the column. However, they do spend some time outside the beads, and eventually, after a full column volume has passed through, they emerge from the column at Vt. The gel

Figure 2 Principles of gel filtration. A mixture of proteins is applied to the column. Only the smallest molecules can penetrate the beads, while the larger molecules pass round the outside of the beads. Intermediate-sized molecules can partially penetrate the beads. As the separation continues, the larger molecules run out ahead of the smaller ones.

Figure 2 Principles of gel filtration. A mixture of proteins is applied to the column. Only the smallest molecules can penetrate the beads, while the larger molecules pass round the outside of the beads. Intermediate-sized molecules can partially penetrate the beads. As the separation continues, the larger molecules run out ahead of the smaller ones.

Figure 3 (A) The elution pattern of a mixture of proteins from a gel filtration column. (B) Plot of the 'elution volume' against the logarithm of the molecular weight. All small molecules emerge at V, all very large molecules earlier at V0, and the values for intermediate-sized molecules fall on a straight line over about a 10-fold increase in molecular weight range. By comparing the results with the elution volumes of proteins of known molecular weight, the molecular weight of an unknown protein can be determined.

Figure 3 (A) The elution pattern of a mixture of proteins from a gel filtration column. (B) Plot of the 'elution volume' against the logarithm of the molecular weight. All small molecules emerge at V, all very large molecules earlier at V0, and the values for intermediate-sized molecules fall on a straight line over about a 10-fold increase in molecular weight range. By comparing the results with the elution volumes of proteins of known molecular weight, the molecular weight of an unknown protein can be determined.

beads have a range of pore sizes, so that intermediate-sized proteins can spend some time inside the beads, but not as much as the smallest proteins. Consequently the emergence volume of each protein is related to size, as shown in Figure 3.

This is also the principle used to separate large molecules from small molecules in general, and is very useful for removing salts from protein solutions. In this case the beads have pores so small that even the smallest proteins cannot penetrate the beads, but salts can.

Gel filtration is a very gentle method; the proteins remain in solution at all times, and need not be exposed to any extremes of pH or salt concentrations. Resolution depends on the relative sizes of the proteins in the starting mixture. Gel filtration will commonly be used at a later stage of purification, when the quantities of sample have been reduced.

There are also membranes available which have pores of suitable size to separate proteins. A force, such as gas pressure, is used to push the liquid, plus smaller molecules through the membrane. The largest molecules are retained, and concentrated behind the membrane. This has application in the relatively crude separation of large proteins from small ones.

Separation by size is the commonest technique in the analysis of proteins, and is described in a later section. Using slab gels, which have pore sizes similar to the beads described previously, proteins are forced by electrophoresis to separate according to their size, the larger ones having more difficulty in finding their way through the gel, and so moving more slowly. This method has been applied preparatively, but only on a relatively small scale and mainly with unfolded, denatured proteins. Normally, one wants to isolate the protein in its native state, as not all proteins can be re-folded in vitro.

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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