Gels on ZAGENO
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Gel electrophoresis is a commonly used method for separating and analyzing biological macromolecules such as DNA, RNA, and proteins based on size and charge. The two most frequent types of gels are agarose and polyacrylamide which can be tailored to your specifications using buffers and other additives.
Agorase Gel is made from non-toxic seaweed polysaccharide polymers and is very easy to prepare and cast. As the pores are not of a uniform size, the gel is not suitable for proteins that are smaller than 200 kDa, but DNA fragments from about 50 base pairs are easily separated. Agarose gels can easily be re-used and stored in a plastic bag in the refrigerator.
Since larger DNA fragments move slower through the matrix than smaller ones, the size of the nucleic acid is determined by how far it has traveled. In order to determine the fragment sizes, you always load a 'ladder' in one of the gel wells. This then provides known fragment lengths within your agarose gel, which in turn tells you the length of your chosen fragments.
To be able to visualize the DNA, you can add a DNA intercalating dye to the gel. Be careful since these dyes may be mutagenic. Commonly used dyes are ethidium bromide and SYBR-dyes.
You generally get a good resolution of 5-10 kb fragments using a 0.7 % agarose gel, whereas small fragments of 0.2-1 kb fragments are better resolved using a 2 % gel. For even smaller fragments, a vertical polyacrylamide gel is more suitable.
Polyacrylamide gels have a much more constant pore size than agarose gels, and the particular pore size is controlled by changing the concentration of polyacrylamide.
Polyacrylamide electrophoresis (PAGE) is most commonly used to separate proteins that have a size ranging from 5-2000 kDa.
After separation, the proteins can be transferred to a membrane and later probed with antibodies and markers. This so-called blotting is what is done when you make a Western blot.
PAGE can be either denatured or native. Denatured PAGE involves the prior denaturation of the macromolecule of interest. This denaturation can be of great benefit since the folding of a protein or nucleic acid influences the mobility through the gel matrix. After denaturation, the mobility of the molecule through the gel is proportional to its length and mass-charge ratio. Commonly used denaturing agents are sodium dodecyl sulfate for proteins (SDS-PAGE) and urea for nucleic acids. DMSO and glyoxal are also used as denaturing agents for RNA.
Native gels are run without disrupting the secondary and tertiary structures of the macromolecule that you are investigating, enabling the size of the folded molecule to be analyzed.
Care should be taken when using acrylamide as it is a potential neurotoxin.
Video & Image Credits
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