CZ:Sandbox: Difference between revisions

From Citizendium
Jump to navigation Jump to search
imported>D. DiJohnson
imported>D. DiJohnson
Line 28: Line 28:


===Limitations===
===Limitations===
The main limitation of agarose gels is their level of resolution.  Agarose gels are not particularly useful for low molecular weight molecules, because properties of agarose don't allow for very fine pores.  Therefore, they are very poor at separating small molecules.
Poly-acrylamide gels are subject to a unique problem known as disulfide scrambling however.  This happens upon denaturing of the peptides.  Disulfide bonds are broken generating sulfide molecules, which can readily form disulfide bonds again.  This is problematic because the peptides sulfide ions can form bonds with the acrylamide gels and change the mobility rate.
Poly-acrylamide gels are subject to a unique problem known as disulfide scrambling however.  This happens upon denaturing of the peptides.  Disulfide bonds are broken generating sulfide molecules, which can readily form disulfide bonds again.  This is problematic because the peptides sulfide ions can form bonds with the acrylamide gels and change the mobility rate.



Revision as of 00:17, 8 November 2010

Gel Electrophoresis

Gel Electrophoresis is a method of separating nucleic acids and peptides using an electric current. The molecules are loaded into a porous gel and an electric current is applied, driving the molecules toward the anode. After the molecules have been separated, various stains and software can be used to visualize and analyze the results.


History

Gel electrophoresis has remained one of the most powerful analytical tools since its development in the early 1900’s. Prior to modern gel electrophoresis, also known as zone electrophoresis because of the distinct zones of material created, electrophoretic techniques produced poorly separated substances in liquid solutions within a tube. Sample movement was monitored by changes in the resulting shadows (3). This format was described by Tiselius and produced only very partially purified samples of the fastest moving particulates (1). The current standard for electrophoresis, including two-dimensional electrophoresis, which still qualifies as zone electrophoresis, relies on movement and containment of samples within a gel matrix according to their individual charge. Two-dimensional gel electrophoresis provides the highest level of separation attainable through gel electrophoresis and can produce over 1000 unique zones (4).

Originally, single dimension gel electrophoresis was used as a method to separate proteins, however with the discovery of DNA, the focus for most of the mid twentieth century was on isolating DNA strands. This trend continued even after the creation of two-dimensional separation techniques by O’Farrell in 1975 (2). While electrophoresis was utilized for both DNA and proteins after the creation of two-dimensional gel electrophoresis, the use for proteins didn’t increase again until after the human genome was sequenced. It was at this point that the focus of research shifted from genomics towards proteomics. With the increased knowledge from the Human Genome Project, the application of two-dimensional gel electrophoresis for cancer research was ideal. Currently, there are many tools which perform powerful separations including LC, HPLC, UHPLC, CE, and GC, however gel electrophoresis remains one of most common due to its ease of operation, high separation resolution, and low cost.


Underlying Principles

Gel electrophoresis works on the basic physical principles that outline the movement of electrons. Gel electrophoresis is possible because all biological molecules have a net charge, whether it‘s positive, negative, or neutral. This fact is essential in the theory behind electrophoresis and is the basis behind the word itself: “electro” refers to a form of energy and “phoresis” originates from the Greek word “to carry”. Biological molecules, DNA, RNA, or peptides, are loaded into a medium, in the form of a porous mesh gel, and a current is applied. Because the molecules have an inherent charge, those with a negative net charge are pulled via the electric current towards the anode. Because many molecules have the same net charge, but vary in size, more specifically mass, and the moving force is constant, the voltage, molecules move according to their charge to mass ratio: z/m, where z is charge and m is mass. By modifying the gel or chemically modifying the molecules themselves, these ratios can be altered, thereby changing which characteristics the molecules are separated by.

Gels

Multiple gel types can be used depending on the application. While these gels can vary in the material they’re made of, the composition ratios of said material, and thickness, all gel types work by creating a network of channels for the molecules to travel through. By adjusting specifics of the gel solution, channels are generated in a variety of sizes. Agarose and poly-acrylamide gels are the two most common gel types. Based on the percentage of either, the diameter of the pores can be adjusted, altering the ability of the strands to travel through it. More agarose or more acrylamide create a tighter mesh and restrict the movement of more molecules.

Almost all polyacrylamide gels make use of two sections of gel. The first portion of the gel, where the sample will be added, is referred to as a stacking gel. Stacking gels are composed of acrylamide, however they consist of a smaller percentage of acrylamide, which creates larger pores. They also contain chloride ions due to a variation in one of the buffers used. By using larger pores and different ions, the sample is quickly integrate into the gel and maintained within a region between fast moving chloride ions and slow moving glycine ions.

Gel Variations

Gel thickness can also be altered. Thicker gels allow more sample to be loaded. If the molecule of interest is in a very low concentration, the ability to add more sample will result in a larger gel band. Gel thickness can be altered in conjunction with the the stain used to generate the desired resolution.


Gradient poly-acrylamide gels are also very common and have two forms. pH gradient poly-acrylamide gels are the main component of Isoelectric Focusing (IEF). For isoelectric focusing, a pH gradient along the gel must be created in order to alter the charge of molecules as they travel through the gel. In pH gradient polyacrylamide gels, molecules are separated according to their isoelectric point (pI), not according to their mass or size.

Gradient poly-acrylamide gels also exist which can use two concentrations or a continuous gradient of acrylamide and a stacking gel. If the sample contains many large molecules, by increasing the percentage of acrylamide throughout the gel, finer separation can be obtained for smaller molecules.

Limitations

The main limitation of agarose gels is their level of resolution. Agarose gels are not particularly useful for low molecular weight molecules, because properties of agarose don't allow for very fine pores. Therefore, they are very poor at separating small molecules.

Poly-acrylamide gels are subject to a unique problem known as disulfide scrambling however. This happens upon denaturing of the peptides. Disulfide bonds are broken generating sulfide molecules, which can readily form disulfide bonds again. This is problematic because the peptides sulfide ions can form bonds with the acrylamide gels and change the mobility rate.

Gel Analysis

One of the most common follow-up solutions is to stain the gel. Several staining methods are available, again, depending on the purpose of the research. A simple Coomasie Blue stain allows for easy visualization of each spot after separation For samples that might be in extremely low concentrations after separation, more powerful stains such as silver stains can detect peptides in concentrations as low as .1ng/mm2 (Bio-Rad Laboratories, Inc., Hercules, CA). For DNA, the most common stain is ethidium bromide, which is only visible under ultra-violet light and is highly carcinogenic. After staining, several software options exist for analyzing spots. These programs are based on algorithms, which can perform a multitude of calculations. Major components of the programs include background subtraction, spot identification, distance analysis, and the ability to search databases for known molecules presenting similar results. Mass spectrometry (MS) is gaining quickly as the final option as many gel protocols allow for easy sample transfer to MS and mass spectrometers become more advance and more common.



References

1. Y. Jin, T. Manabe, Analysis of PEG-fractionated high-molecular-mass proteins in human plasma by non-denaturing micro 2-DE and MALDI-MS PMF. Electrophoresis 2009, 30. 3613-3621, DOI: 10.1002/elps.200900191.

2. P. H. O'Farrell, High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 1975, 250. 4007-4021.

3. P. G. Righetti, Electrophoresis: The march of pennies, the march of dimes. Journal of Chromatography A 2005, 1079. 24-40.

4. O. Vesterberg, Separation of Proteins in Blood Plasma and Serum by Isoelectric Focusing and Two-Dimensional Electrophoresis. National Inst. Occupational Health 1988, 48. 99-101, DOI: doi:10.1080/00365518809168524.