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. At this pH, glycine is predominately negatively charged, forming glycinate anions.
When an electric field is applied, glycinate anions hit the pH 6. That means they move slowly through the stacking layer toward the anode due to their lack of charge. By contrast, the Cl- ions from the Tris-HCl in the gel move at a faster rate towards the anode. When the Cl- and glycine zwitterions hit the loading wells with your protein samples, they create a narrow but steep voltage gradient in between the highly mobile Cl- ion front leading ions and the slower moving, more neutral glycine zwitterion front trailing ions.
The electromobilities of the proteins in your sample are somewhere in between these two extremes, and so your proteins are concentrated into this zone and herded through the stacking gel between the Cl- and glycine zwitterion fronts. What happens to glycine zwitterion in the resolving layer? It gets real negative, real fast. When the Cl- and glycine zwitterion fronts hit the resolving layer at a pH of 8. They are no longer predominately neutral and take off towards the positively charged anode as glycinate anions.
Unaffected by polyacrylamide, they speed past the protein layer, depositing the proteins in a tight band at the top of the resolving layer. What happens to the proteins in the resolving layer? They slow way down and start to separate. The proteins moved more easily through the stacking layer because of the low percentage of acrylamide.
Now that they are starting into the resolving layer which has a higher percentage of acrylamide, they have to slow down. All buffers have different solutions to enable optimum gel electrophoresis or protein transfer with their corresponding gel chemistry. Once the protein reaches the resolving gel , the pH changes from 6.
As pH increases, the N-terminal amino groups are deprotonated. Amino acids and proteins are more negatively charged at equilibrium than in stacking gel. As a result, glycine moves faster than proteins. In SDS - PAGE , the use of sodium dodecyl sulfate SDS , also known as sodium lauryl sulfate and polyacrylamide gel largely eliminates the influence of the structure and charge , and proteins are separated solely based on polypeptide chain length.
Thus, when a current is applied, all SDS -bound proteins in a sample will migrate through the gel toward the positively charged electrode. What is discontinuous gel electrophoresis? Category: medical health infertility. Discontinuous electrophoresis colloquially disc electrophoresis is a type of polyacrylamide gel electrophoresis. It was developed by Ornstein and Davis. This method produces high resolution and good band definition.
It is widely used technique for separating proteins according to size and charge. What is the difference between stacking gel and separating gel? What is stacking gel and resolving gel? What is slab gel? The Cl- ions from Tris-HCl on the other hand, move much more quickly in the electric field and they form an ion front that migrates ahead of the glycine.
The separation of Cl- from the Tris counter-ion which is now moving towards the anode creates a narrow zone with a steep voltage gradient that pulls the glycine ions along behind it, resulting in two narrowly separated fronts of migrating ions; the highly mobile Cl- front, followed by the slower, mostly neutral glycine front.
All of the proteins in the gel sample have an electrophoretic mobility that is intermediate between the extreme of the mobility of the glycine and Cl-, so when the two fronts sweep through the sample well, the proteins are concentrated into the narrow zone between the Cl- and glycine fronts.
This procession carries on until it hits the running gel, where the pH switches to 8. At this pH the glycine molecules are mostly negatively charged and can migrate much faster than the proteins. So the glycine front accelerates past the proteins, leaving them in the dust. The result is that the proteins are dumped in a very narrow band at the interface of the stacking and running gels and since the running gel has an increased acrylamide concentration, which slows the movement of the proteins according to their size, the separation begins.
Gel wells are around 1cm deep and you generally need to substantially fill them to get enough protein onto the gel. So in the absence of a stacking gel, your sample would sit on top of the running gel, as a band of up to 1cm deep. Rather than being lined up together and hitting the running gel together, this would mean that the proteins in your sample would all enter the running gel at different times, resulting in very smeared bands.
So the stacking gel ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands.
Once the proteins are in the running gel, they separate because higher molecular weight proteins move more slowly through the porous acrylamide gel than lower molecular weight proteins. The size of the pores in the gel can be altered depending on the size of the proteins you want to separate by changing the acrylamide concentration.
Typical values are shown in Table 1 below. For a broader separation range, or for proteins that are hard to separate, a gradient gel , which has layers of increasing acrylamide concentration, can be used. If you have any questions, corrections or anything further to add, please do get involved in the comments section!
Originally published on September 18, Revised and updated August
0コメント