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| Chemical analysis in biology is pretty
complicated, owing to the size and complexity of the molecules involved in
most of the processes. Think about the stages of respiration and
photosynthesis - for almost every conversion of relatively simple
carbohydrates, there are enzymes, coenzymes, and carriers involved -
mostly
huge, intricate molecules whose activity depends upon subtle differences
in sequences and conformation. And all of these molecules have been
assembled in the cells and mixed into the cytoplasmic soup - how can a
researcher ever pick out just a single molecule to analyze? |
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Separation of Proteins by Electrophoresis |
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One fairly simple method to sort through a mixture of proteins takes advantage of the differences in size and charges among the molecules. Electrophoresis uses a support medium of some type - starch, paper, various types of gels - to both float the mixture and support an electric field. The electric field is engaged for a set amount of time, during which the proteins migrate from the positive end of the field (where the mixtures are placed) to the negative end, depending upon the various charges of the amino acids in each protein (and the interactions with the charged medium). The proteins have been denatured to make them interact better with the medium, as well to shut down the activity of enzymes that could interfere with the process. A protein which is overall more positively charged will migrate farther toward the negative pole during the process. But that's not the only factor that affects how far the protein moves - smaller proteins will pass through the medium faster than large ones. When the process ends and the electric field is turned off, the medium gets stained to make the migrated proteins show up. This process can be used to isolate proteins for further analysis, as well as to find differences in proteins in two near-identical systems, such as closely-related organisms. It has been used to find differences between normal disease organisms and organisms that have developed drug resistances. If a mutated protein is involved in the resistance, it may be different enough from the normal variety to move to a different place during electrophoresis. Electrophoresis can also be used to analyze single molecules, of DNA as well as proteins. If a batch of a single protein is treated with enzymes that clip the sequence wherever a particular type of peptide bond exists, the resulting bits can be separated by electrophoresis. Each bit can be analyzed in a similar way, and rough sequences can be built, as well as isolating critical differences between very similar proteins. A high-tech variation on this basic idea is
mass
spectroscopy, which uses dispersal of materials as gases and
movement through varied-length magnetic fields (the longer the track, the
more separation between similar-but-different molecules can occur).
This can be used to separate and identify a wide variety of organic
molecules, and can be sensitive to tiny
differences. The technique
was originally called atomic mass spectroscopy and was used to
distinguish
isotopes
from one another. A very good ( = expensive) mass spectrometer can identify a particular molecule, different
by just an atom or even a neutron, by how long it takes to get from the
intake to the meter. |
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Online Introduction to Biology (Advanced)
Copyright 2004 - 2008, Michael McDarby.
Reproduction and/or dissemination without permission is prohibited. Linking to the site is fine.