Separating individual proteins from complex protein mixtures can be achieved by gel electrophoresis in poly acrylamide gel electrophoresis, otherwise known as page proteins migrate in response to an electrical field through pores in the gel matrix. The combination of gel pore size and protein charge size and shape determines the migration rate of the protein. In this video, we will demonstrate a method for electrophoretic separation of proteins.
Hi, I'm Dr.Bobul Chakravarti from the Proteomic Center of the Keck Graduate Institute of Applied Life Sciences. Today I'm going to show you a procedure for doing 1D gel electrophoresis. So let's get started.
To begin, assemble the glass plate sandwich of the electrophoresis apparatus according to manufacturer's instructions using two clean glass plates and two 1.5 millimeter spacers. Now lock the glass plate sandwich to the casting stand. Next, prepare the separating gel solution which has been described in current protocols.
Add the specified amount of 10%ammonium per sulfate and teed to the solution and stir gently to mix. Due to the high concentration of acrylamide, degassing is not necessary. Using a 10 milliliter disposable plastic pipette, immediately apply the separating gel solution to the sandwich along an edge of one of the spacers until the height of the solution between the glass plates is about 11 centimeters.
Next, use another pipette to slowly cover the top of the gel with a layer approximately one centimeter in thickness of water saturated isobutyl alcohol or distilled water. Do this by gently layering the water against the edge of one, and then the other of the spacers allow the gel to polymerize 30 to 60 minutes. At room temperature, the water overlay ensures that the acrylamide gel polymerizes into a straight line.
Once the gel polymerizes, we're ready to pour the stacking gel to pour the stacking gel. First, pour off the layer of distilled water, then rinse with one x tris CL in SDS at a pH of 8.8. Now prepare the stacking gel solution as indicated in current protocols.
Next, using a plastic pipette, allow the stacking gel solution to trickle slowly into the center of the sandwich along an edge of one of the spacers until the height of the solution in the sandwich is about one centimeter from the top of the plates. Be careful not to introduce air bubbles into the stacking gel. Once the gel is poured, insert a 0.75 millimeter Teflon comb into the layer of stacking gel solution.
Allow the stacking gel solution to polymerize 30 to 45 minutes At room temperature, we are now ready to prepare the protein samples. To begin the protein sample preparation. Dilute a portion of the protein sample to be analyzed at a one-to-one ratio with two XSDS sample buffer.
For dilute protein solutions, consider using a five to one protein to six times SDS sample buffer mixture. If the sample is a precipitated protein pellet, dissolve the protein in 50 to 100 microliters of one XSDS sample buffer. Now heat the samples for three to five minutes at 100 degrees Celsius in a sealed screw cap, micro centrifuge tube in a water bath.
Next, carefully remove the Teflon comb without tearing the edges of the poly acrylamide wells. After the comb is removed, rinse wells with one XSDS electrophoresis buffer. Then using a pipette, fill the wells with one XSDS electrophoresis buffer.
Once the wells are rinsed and filled with buffer, attach the gel sandwich to the upper buffer chamber. Following manufacturer's instructions, fill the lower buffer chamber with the recommended amount of one XSDS electrophoresis buffer. Next, place the sandwich attached to the upper buffer chamber into the lower buffer chamber.
Now completely fill the upper buffer chamber with one XSDS electrophoresis buffer so that the sample wells of the stacking gel are filled with buffer. The gel is now ready to be loaded using a 25 or 100 microliter auto pipetter with gel loading tips, load protein sample into one or more wells by carefully applying the sample as a thin layer at the bottom of the wells. Load control wells with molecular weight standards.
Also add an equal volume of one XSDS sample buffer to any empty wells to prevent spreading of adjoining lanes. We are now ready to start the gel electrophoresis. To run the gel, connect the power supply to the cell and run it at 30 milli piers of constant current for a slab gel 1.5 millimeters thick.
After the menthol blue tracking dye has reached the bottom of the separating gel, disconnect the power supply. Now that the gel has finished running, discard the electrophoresis buffer and remove the upper buffer chamber with the attached gel sandwich. Orient the gel so that the order of the sample wells is known.
Then remove the sandwich from the upper buffer chamber and lay the sandwich on a sheet of absorbent paper or paper towels. Next carefully, slide one of the spacers halfway from the edge of the sandwich along its entire length. Use the exposed spacer as a lever to pry open the glass plate, exposing the gel carefully.
Remove the gel from the lower plate. Cut a small triangle off one corner of the gel so the lane orientation is not lost during staining and drying. The gel electrophoresis procedure is now complete.
The gel can now be processed using various protein detection procedures. We have just shown you how to separate proteins using gel electrophoresis. So that's it.
Thanks for watching and good luck with your experiment.
