I'm planning a science project about gel electrophoresis, and I'd like to know if there are any measurable, quantifiable (for example, things I could represent in a graph or chart) differences between the behavior of protein and DNA during agarose gel electrophoresis. Detailed inputs and useful links are greatly appreciated.
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$\begingroup$ Proteins are usually not run on agarose gels since the pores of the gel are too big to have a good separation. A 1% agarose gel has pores of around 150nm, poly acrylamide (which is usually used) have 3-6nm depending on the degree of polymerisation and gel strength. You will only use agarose gels for really high molecular weight proteins otherwise you won't see much separation of the sample. DNA samples have such a high molecular weight and can thus be separated on agarose gels. $\endgroup$– Chris ♦Commented Jan 16, 2014 at 20:02
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$\begingroup$ @Chris One of the requirements of the project is to run several simultaneous experiments while changing one variable only (in this case, it's the protein/DNA). I'm separating proteins in dyes, which would explain the use of agarose. $\endgroup$– Dan HumphreyCommented Jan 16, 2014 at 20:06
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$\begingroup$ What do you mean by "separating proteins in dyes"? Another thing: While you can't separate Proteins nicely on agarose, you can use low percentage polyacrylamide gels to separate DNA. $\endgroup$– Chris ♦Commented Jan 16, 2014 at 20:12
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$\begingroup$ @chris Excuse the typo. I meant that electrophoresis will be run on food coloring dyes... you know... which are made of proteins $\endgroup$– Dan HumphreyCommented Jan 16, 2014 at 20:21
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1$\begingroup$ As already mentioned, you don't use agarose for proteins, they're pretty much exclusively run on polyacrylamide gels, typically as an SDS-PAGE. I've never seen anyone run an agarose gel of a protein. $\endgroup$– Mad ScientistCommented Jan 16, 2014 at 21:16
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1 Answer
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There are two important properties that determine how a molecule will move during gel electrophoresis:
- size and shape of the molecule
- charge of the molecule
You're applying an electric field, and all molecules will move according to their charge. The gel itself hinders the movement of larger molecules. This means that small molecules will move further than large molecules.
Nucleic acids like RNA and DNA have a uniform negative charge from the phosphate backbone. This means that they will generally separate according to their size and shape (plasmids can be present in different forms like supercoiled, circular or linearized).
Proteins have different charges, some have a positive charge at the pH typically used for gel electrophoresis, some have a negative charge, and some have no charge at that pH. So if you would simply run proteins on a gel, some would move backwards, some forward and some not at all.
To make the proteins separate according to their size we use the detergent SDS to denature the proteins, and to give them a uniform charge (SDS is negatively charged). Proteins are also run on polyacrylamide gels and not agarose as the size of the pores in agarose is a bad fit for the typical sized of proteins.
While I've never seen anyone using agarose for proteins, it seems that it is possible and sometimes used for larger proteins. I found one paper describing a protocol, they used very high concentrations of agarose in the range of 4-5%. I suspect that they won't handle well, but you should try that out yourself. If the proteins you're examining are a bit larger, using agarose should be possible.
answered Jan 17, 2014 at 9:51
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$\begingroup$ Just a small addition.. Agarose pore size is huge for most proteins. However, agarose gel is used for analyzing antibody-antigen interaction. The complexes that form are huge and can be separated by AGE. See rocket immuno-electrophoresis $\endgroup$– WYSIWYGCommented Jan 18, 2014 at 5:42