Proteomics/Protein Separations- Electrophoresis/Gel Electrophoresis/Types of Gel Electrophoresis
Gel electrophoresis is a group of techniques used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point as they are forced through a gel by an electrical current. Gel electrophoresis is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization. (Wikipedia).It is based on the principle that,when charged molecules are placed in an electric field, they migrate toward either the positive or negative poledepending on their charge. The phosphate backbone in nucleic acids render them a consistent negative charge, so they migrate toward the anode. But proteins can have either a net positive or net negative charge.
GE of Nucleic acids
In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the natural negative charge due to the sugar phosphate backbone of a DNA molecule. Double-stranded DNA fragments are normally long stranded molecules, therefore their migration through the gel is relative to their size of DNA molecules and amount of bases. Single-stranded DNA or RNA tend to fold into tertiary structure due to their wearer single backbone and travel through the gel in a complicated manner. DNA can be denatured by breaking hydrogen bonds and renatured to provide easier and more accuarate results frm the gel.
GE of Proteins
Proteins vary on many different levels in comparison to DNA. Protein can have different charges and complex shapes, primary, secondary, teritary, and quaternary structure that make migration through the gel have extremelyt different rates when placing a negative to positive EMF on the sample.Proteins are commonly separated using polyacrylamide gel electrophoresis (PAGE). PAGE can be used to purify proteins prior to other proteomics techniques or to analyze to get information on the mass, charge, or presence of a protein.SDS-PAGE is the most widely used electrophoresis technique for separates proteins.Due to these complex structures proteins are usually denatured, or broken down to simple primary structures in the presence of a detergent such as sodium dodecyl sulfate (SDS)which coats the proteins with a negative charge.This allows the protein to have an overall negative charge and allow for proper migration. The amount of SDS bound and used is relative to the size of the protein,thus this method separates proteins mainly based on mass. Two-dimensional PAGE (2-D PAGE) separates proteins by isoelectric point in the first dimension and by mass in the second dimension.Native PAGE separates proteinsby mass/charge ratio without denaturing them.
Electrophoreseis is usually carried out within a matrix or gel made of agarose or polyacrylamide. These gels are chemically inert, so they will interfere little with the molecules. The sample is loaded in the gel;in wells for nucleic acids separation. Agarose is a polysaccharide extracted from seaweed. Agarose gels have a large range of separation depending on the concentration.The higher the concentration the smaller the pore size will be.Polyacrylamide is a cross-linked polymer of acrylamide.To avoid inhibition of polymerization by oxygen, they are poured between glass plates to mage gel slabs.Low concentration of polyacrylamide or less cross linking results in gels with large pores. Standard protein gels are typically composed of two layers, ahe top-most layer called the stacking gel and a lower layer called separating or resolving gel.The stacking layer contains a low percentage of acylamide and low pH , while the acrylamide concentration of the separating gel varies according to the samples to be run and pH is higher.The difference in pH and acrylamide concentration at the stacking and separating gel provides better resolution and sharper bands in the separating gel.Acrylamide is a neurotoxin and therefore person handling the gel should wear gloves to avoid contact with it. The polyacrylamide gels have a high resolving power compared to agarose gels.Dependent on the substance being run, the composition, pourous nature, and percentage of gel will be varied to provide proper separation. This separation using acrylamide is normally done on proteins, DNA, RNA, oligonucleotides and other smaller molecules due to its less pourous nature. Using different concentration and compositions of aryclamide provide a thicker or thinner matrix which will separate based on size and charge. Separating larger nucleic acids is normally done with agarose gels.Again cases of concentration and composition can vary the results of the distance a substance travels on a gel.
The samples are loaded in the wells in the gel covered with buffer. The elctrophoresis buffer provide uniform pH and provide ions to support conductivity. When an electric current is applied, the molecules move through the matrix at different rates, towards the anode if negatively charged or towards the cathode if positively charged. (Wikipedia) The voltage as well as the time depend on the size of the gel and the type of sample. Caution must be taken to not run the samples too long or the samples may run off the gel. Caution must also be taken with the voltage, voltage too high may burn the gel or damage the samples.
When the gel electrophoresis has completed its run the gel is stained to allow for the bands of sample to be seen and analyzed. Stains that are normally used are Ethidium Bromide, silver, or coomassie blue dye depending on the sample. The gels are cafefully removed and placed directly into staining solution where it will sit overnight to allow for proper staining. Once fully stained bands should appear if samples were run correctly along with a molecular mass marker be placed on one or both ends of the gel to provide proper analysis.This method of protein separation and identification is useful because very little protein is needed to determine a difference in a protein. Distinct spots can be found with as little as 0.1 mg of protein when stained with Coommasie blue and even less (0.02 mg), is need when silver staining. Other methods of staining are Fluorescent dyes, and zinc or copper staining.
Visualization can be done by the naked eye as well as under a UV light to provide easier visualization.If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactive atoms, an autoradiogram can be recorded of the gel
Bands are then compared to those of the known molecular mass marker on the sides of the gel to determine the approximate mass of the substance as well as separate out bands. A sample that has several bands coming from a single sample can indicate an impure sample. Further analysis can be done on proteins by removing the separted sample from the gel by cutting the band out and using In-gel digestion to remove the protein from the gel for further analysis.Some drawbacks to this technique are that it is time-consuming and the purity of your protein sample will affect your results.