Fractionation and Separation of Molecules
Using Gel Filtration Chromatography
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Angela Duff |
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Neuronal activity is determined by the ion channels in the neuronal cell membrane and how easily they can be stimulated to open. Ion channels are proteins found in the cell membranes of neurons which permit certain ions to pass through the membrane. Common ion channels include those for sodium, potassium, chlorine, and calcium. Channels can be opened by altering membrane voltage or by chemical stimulation. The amount of stimulus necessary to open certain ion channels can be modulated by hormones and proteins.
Dr. Romano's lab has found that a molecule in a certain tissue extract modulates the activity of an ion channel, and it is our goal to isolate that molecule. This research is pertinent because no substance is yet known to modulate the activity of this particular ion channel. The tissue estract is a very complex mixture of molecules. Purification of a single particle requires separation of the extract into subfractions based on size, charge, lipophilicity, and other characteristics. This summer, I have been working on separating substances by size using gel filtration chromatography.
Gel filtration chromatography allows for precise separation of materials by size. The setup consists of a glass cylinder filled with gel, a pump, and a fraction collector. The tissue extract is placed on top of the gel. Buffer solution is then pumped through the column, forcing the material through the gel to the fraction collector. The fraction collector is a machine that drips the material into different test tubes as the solution leaves the column. This can be done using a tube that moves over a tray of test tubes, or a tray of tubes which moves under the three-way valve located at the bottom of the column.
The resulting fractions contain molecules that have been separated by molecular size. The larger molecules are the first to exit the column, and the smaller molecules come out later. This is due to the gel in the column. The gel is composed of small beads that the smaller molecules passing through the gel enter. Because the small molecules enter the gel beads, they must pass through the entire volume of the column. Larger molecules that cannot enter the beads only have to pass through the buffer outside the beads. The fractions are analyzed to determine the amount of different sized proteins found. The absorbance of certain wavelengths of light, specifically 215nm and 280nm, is measured.
I ran columns with solutions of molecules of known molecular weight and with samples of the tissue extract. I first used a P6 gel, which showed one large peak where the majority of the high molecular weight material was expelled and a smaller peak where the low molecular weight material was expelled. I then ran the same material in a P150 gel that gave better resolution of the first peak. Several different peaks were visible in the larger peak.
In order to assay the different fractions, Dr. Romano will cause frog oocytes to express the channels in their cell membranes. This is done by injecting the eggs with mRNA. The channels would then be stimulated in the presence and absence of varying fractions from the column. Unfortunately, these assays have been delayed due to problems with the oocytes.
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