The strength of the gel allows easy handling. Polyacrylamide gel electrophoresis of SDS-treated proteins allows researchers to separate proteins based on their length in an easy, inexpensive, and relatively accurate manner. Custom MHC tetramer services. Principle and method of the experiment Webinar FAQ.
The principle. A comb is used to make wells lanes to load samples. Use an appropriate comb depending on the sample size. Example: Use an 8-lane comb for 7 samples and molecular weight markers. Thoroughly clean the glass plates with ethanol, and assemble the gel casting mold. Pour acrylamide solution for a separating gel. Overlay with water to prevent contact with air oxygen , which inhibits polymerization. Allow acrylamide to polymerize for minutes to form a gel.
Remove the overlaid water. BME breaks up disulfide bonds in the proteins to help them enter the gel. Glycerol adds density to the sample, helping it drop to the bottom of the loading wells and to keep it from diffusing out of the well while the rest of the gel is loaded. Bromophenol Blue is a dye that helps visualization of the samples in the wells and their movement through the gel. Sample loading buffer is also known as Laemmli Buffer, named after the Swiss professor who invented it around What is in the gels?
Although the pH values are different, both the stacking and resolving layers of the gel contain these components. Tris and SDS are there for the reasons described above. The Cl- ions from the Tris-HCl work with the glycine ions in the stacking gel. Again, more to come on that. What is in the gel that causes different sized protein molecules to move at different speeds?
Pore size. When polyacrylamide is combined in solution with TEMED and ammonium persulfate, it solidifies, effectively producing a web in the gel. It is through this web that the linearized proteins must move. When there is a higher percentage of acrylamide in the gel, there are smaller pores in the web. This makes it harder for the proteins to move through the gel. When there is a lower percentage, these pores are larger, and proteins can move through more easily.
Why are there different percentages of acrylamide in gels? To optimize the resolution of different sized proteins. Different percentages of acrylamide change the size of the holes in the web of the gel. Larger proteins will be separated more easily in a gel that has a lower percentage of acrylamide — because the holes in the web are larger.
The reverse is true for smaller proteins. They will resolve better in a gel with a higher acrylamide percentage because they will move more slowly through the holes. Small proteins will fly through a low percentage gel and may run off the end of the gel. WHAT are there two layers in the gel? The stacking layer and the resolving layer. The top stacking layer has a lower percentage of acrylamide and a lower pH 6.
There is discontinuity not only between the gels different pH values and acrylamide amounts , but also between the running buffer and the gel buffers. The running buffer has different ions and a different pH than the gels.
WHY are there two layers in the gel? They have different functions. The stacking layer is where you load your protein samples. The purpose of the stacking layer is to get all of the protein samples lined up so they can enter the resolving layer at exactly the same time.
When you load a gel, the wells are around a centimeter deep. If your samples entered the resolving layer this spread out, all you would see is a big smear. The resolving layer then separates the proteins based on molecular weight.
How does the stacking layer do its job? Low acrylamide content and low pH. The low percentage of acrylamide in the stacking layer allows for freer movement of the proteins and helps them line up to enter the resolving layer together. The lower pH allows glycine to be in its zwitterionic state.
Wait — did you just sneeze? I said glycine is a zwitterion at pH 6. A lot. It is the key to the discontinuous buffer system. It is the ionic state of glycine that really allows the stacking buffer to do its thing. The charge of its ion is dependent on the pH of the solution that it is in. In acidic environments, a greater percentage of glycine molecules become positively charged.
At a neutral pH of around 7, the ion is uncharged a zwitterion , having both a positive charge and a negative charge. At higher pHs, glycine becomes more negatively charged.
Glycine is in the running buffer, which is typically at a pH of 8. They find themselves in a much more relaxed, uniform electric field where they can chill out a bit. Move at their own pace.
After pouring the running gel, carefully overlay it with ethanol or another imiscible liquid. This will give you a nice flat surface. Also, since polymerization of acrylimide is inhibited by oxygen it will speed up polymerization.
For the mini-gels we run the minimum protein loading per well single band is 0. If you exceed amount this your gel will look like crap. KCl causes SDS to precipitate. If you samples contain KCl you should dilute them or methanol precipitate them and resuspend them in 1X sample buffer. This will help the gel run a little less anomalously. If your sample buffer turns yellow, it is at the wrong pH.
Add NaOH or. Safety Notes: - Acrylimide is extremely toxic, causing central nervous system paralysis. It can be absorbed through unbroken skin. If skin comes in contact with acrylimide solution or powder, wash immediately with soap and a lot of water. Unpolymerized acrylimide should be polymerized with excess catalyst and disposed of with solid waste. It goes bad after a week or two in the refrigerator. It can be disposed of by dilution with water and pouring down the sink. A bottle should be good for about a year, maybe longer.
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