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Protein Electrophoresis

Protein Electrophoresis

Summary

Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) is a very common technique used for the analysis of complex mixtures of polypeptides. It has great resolving power, is fast, and is suitable for acidic or basic PIK proteins. The last reason is that the protein reacts with SDS, which binds the protein at a ratio of approximately 1∶4∶1 (SDS:protein, w/w) and imparts a negative charge to the SDS–protein complex. The charged complexes move towards the anode when placed in an electric field, and separate based on the difference in charge and size. SDS-PAGE is most commonly used to estimate the molecular weight of a protein, but estimates are approximate (called “apparent molecular weight”) and are sometimes prone to marked error.

For example, a disproportionately large increase in apparent molecular weight may result from covalent phosphorylation of proteins (1), or artificial entrapment of phosphoric acid (2). Most of the SDS-PAGE designs use a “stacking gel”. Such a system enables the concentration of samples from a comparatively large volume within the gel to very small areas, thus giving narrow bands of different proteins, which are then optimally resolved.

The principle involved in this protein concentration (or “stacking”) is isotacophoresis. This is set up by creating a stacking gel on top of the “separating gel”, which is of a different pH. The sample is mounted on the stacking gel and when electric field is applied, negatively charged complexes and small ions move towards the anode.

Protein Separation Methods
The following is a quick review of some common methods used for protein separation.
:
SDS-PAGE (SDS-polyacrylamide gel electrophoresis) Mainly
separates proteins based on molecular weight, as opposed to charge (that is, ‘swamp out’
occurs) excess of protein-bound SDS) or folding (
Proteins are extensively denatured in SDS).

Protein sequencing
Protein sequencing

What is protein electrophoresis?

Protein electrophoresis is a standard laboratory technique by which charged protein molecules are moved through a solvent by an electric field. Both proteins and nucleic acids can be separated by electrophoresis, which is a simple, fast and sensitive analytical tool. Most organic molecules carry a net charge at any pH other than their isoelectric point and will migrate at a rate proportional to their charge density. The mobility of a molecule through an electric field will depend on the following factors: field strength, net charge on the molecule, size and shape of the molecule, ionic strength, and properties of the matrix through which the molecule migrates (eg, viscosity, bore size). Polyacrylamide and agarose are two support matrices commonly used in electrophoresis. These matrices act as a porous medium and behave like a molecular sieve. Agarose has a large pore size and is suitable for separating nucleic acids and large protein complexes. Polyacrylamide has a small pore size and is ideal for separating most proteins and small nucleic acids.

Several forms of polyacrylamide gel electrophoresis (PAGE) exist, and each form can provide different types of information about the protein of interest. Reducing and reducing sodium dodecyl sulfate PAGE (SDS-PAGE) with a closed buffer system is the most widely used electrophoresis technique and separates proteins primarily by mass. Nondenaturing PAGE, also called native-PAGE, separates proteins according to their mass/charge ratio. Two-dimensional (2D) PAGE separates proteins by native isoelectric point in the first dimension and by mass in the second dimension.

 

 

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