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gel rig info
gel simulator
constructing a standard curve
Experiment 4
Quantitation & Electrophoresis of Proteins
The Support Matrix
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Many different support matrices are available for separation of proteins and nucleic acids. In this lab you will use polyacrylamide, one of the most commonly used support matrices, to separate the histone proteins. Polyacrylamide makes a porous gel which acts as a sieve by retarding or, in some cases by completely obstructing, the movement of certain size macromolecules while allowing smaller molecules to migrate freely through the pores. In forming the polyacrylamide gel, acrylamide monomers polymerize into long chains that are covalently linked by the crosslinker N, N'-methylene-bis-acrylamide (bis for short). |
The pore size in a polyacrylamide gel can be altered in either of 2 ways:
Polymerization is typically accomplished using a chemical reaction. The most common chemical method is by using ammonium persulfate and the quaternary amine, N, N, N', N' - tetramethylethylenediamine (TEMED) which are the initiator and the catalyst respectively.
Gloves should be used throughout the process of handling polyacrylamide gels! Unpolymerized acrylamide is deadly. Theoretically, all of the acrylamide should be polymerized by the time the gel is ready to be loaded, but care should be exercised in case any residual unpolymerized acrylamide is present on the gel rig.
Acrylamide gels can be poured by the user or purchased precast. The electrophoresis system we will be running uses gels that are precast in a plastic cassette. In the cassette, the gel is polymerized between two plates separated by plastic spacers. A comb inserted between the two plates creates wells in the gel for loading the samples. Before use, the comb is carefully removed and the cassette is locked into place in the gel rig..
The rigs we use has positions to hold two gels, or if only one gel is to be run, a plastic spacer the same size as a gel is placed on the unused side. After the rig is assembled, running buffer is poured into the inner and outer buffer tanks. The inner tank is filled until the level of buffer comes into contact with the top of the gel.
In order to set up the electric field that causes the movement of the negatively charged protein micelles through the gel, a complete circuit must be set up through the gel rig. Because of the compactness of the gel rig, it is a bit difficult to trace the complete path of current through the gel. Study the two diagrams below to see how the circuit is completed.
The wells are loaded by squirting the protein samples into them with a special capillary pipette tip that is narrow enough to fit between the plastic plates. If you are loading more than 20 ul of sample, you will need to use the tip with a P200. While looking down at the top of the rig, carefully insert the pipette tip between the two plates as shown below.
After you have the pipette tip between the plates, look through the front of the rig so that you can make sure that the pipette tip is entering the desired well. The protein is mixed with a loading dye that is more dense than water, so that the sample will sink to the bottom of the well. When the tip is in place, gently expel the sample into the well. Remove the pipette tip from the well before releasing the plunger of the pipetter.
After the rig is loaded, a special lid is placed on the rig. The wires attached to the lid connect with the terminals of the rig when the lid is on. This safety interlock system reduces the chance of electrical shock by disconnecting the system from the power supply when the lid covering the tanks is removed. The voltage is adjusted to an appropriate level and the proteins begin to move downward through the gel.
Because the voltage is applied to the gel across the top and bottom, the
electric field in the gel is oriented vertically. Thus, the charged
proteins are driven to move downward through the gel directly under the well in
which they were loaded. The path that the proteins take is called a lane.
The size standards we use are prestained, so you can see them moving in the gel
image above. Their location can be used as a guide to
know when the voltage should be turned off. The unknown proteins are not
visible as they move through the gel.
After the power is turned off, the gel is removed by prying apart the two sheets
of plastic. It is placed in a tray containing Coomasie blue and is agitated
for a period of time to allow the dye to bind with the protein in the gel.
It is then destained by soaking in buffer to remove the unbound dye from the
gel. The stained proteins appear as bands on the gel. The gel is
photographed and then analyzed to determine the standard curve and sizes of the
unknown proteins.
Gel simulator (courtesy of http://webphysics.davidson.edu/applets/biogel/biogel.html)
You can experiment with varying the gel density and electric field (i.e. through changing the voltage) using the simulator below. This simulator mimics an agarose gel separating eight DNA fragments. The fragment sizes (in kilobasepairs) are entered in the boxes at the top. The agarose concentration and electric field are adjusted using the sliding controls at the bottom. When you press compute, the simulator shows how far the bands would move from left to right during a fixed run time. (The run time can not be varied.) You will be asked to play with this simulator for the prelab.
On a particular gel, we do not know the relationship between protein weight and the distance traveled. Thus a standard curve must be constructed, as discussed in the lab manual. Proteins of known molecular weight are electrophoresed alongside with polypeptides of unknown molecular weight. Under most conditions, the electrophoretic mobility of a proteins is inversely proportional to the log of their molecular weight. The standard curve for electrophoresis is constructed by plotting the log of molecular weight of known proteins (y-axis) versus the distance migrated (x-axis) measured from the sample well.
