The two layers have different functions: the stacking gel is needed to concentrate/pack all the proteins in one band, so that they will start migrating in the running gel all at the same time. The running gel allows to separate the proteins in the sample based on their molecular weight
The two layers have different functions: the stacking gel is needed to concentrate/pack all the proteins in one band, so that they will start migrating in the running gel all at the same time. The running gel allows to separate the proteins in the sample based on their molecular weight
As the other name suggest the stacking gel is where the protein sample loaded is stacked and in the separating or running gel the protein migrate according to their molecular weight, lower the mol. wt faster it will move. and if there is no stacking gel the proteins will not resolve properly.
The stacking gel has large sized pores that allow the proteins to migrate freely and get stacked at the interface between stacking gel and running gel (hence the name ‘stacking’ gel). Purpose of this is to make sure that the proteins start migrate from the same level, since they are separated based on their mass (the effect of their charge is overcome by SDS’s high negativity). The sharpening of band which you can appreciate visually while the proteins migrate in stacking gel is due to the phenomenon called ‘isotachophoresis’. Due to isotachophoresis the migrating proteins get sandwiched tightly between the glycinate ions and chloride ions that migrate along, which makes the proteins to get aligned in sharp band. Hope this helps (Reference: Wilson and Walker, Principles and techniques of biochemistry and molecular biology, Pg:407)
Stacking gel has high acrylamide concentration and high voltage is applied to it thus it helps the proteins to come in one race line before starting the race.
Resolving gel is the actual track where proteins run according to their molecular weight.
I also encountered this same difficulty. I understood the concept of the role of stacking gel involved in but not fully. I would be highly glad if anyone could explain me a little more.
The question that comes in my mind is: What will be the outcome if only resolving gel is used ?
If the answer is a smear. Then, what resulted in the smear?
Thank you.
While running an SDS-PAGE gel we use 3 buffers, Tris- Gly (8.3), Tris-Cl (pH 6.8) & Tris-Cl (8.8). The Tris-Cl buffers are present in the stacking & resolving gels respectively. The Tris-Gly is the buffer used for running the apparatus. In Tris-Gly @ pH 8.3 the glycine exists as a –ve charge and moves towards the positive electrode. So this –vely charged Gly enters the stacking gel. In the stacking gel, the pH changes to 6.8 where Gly exists in zwitter-ionic form. Now Gly moves slowly but the Cl- (from Tris-Cl) moves fast and reaches the interface of resolving and stacking gel first. So in the stacking gel the Cl- forms the leading front & glycine forms the back-front with proteins in between these fronts. Now apart from the electric field provided by the electrodes, another electric field develops due to differential movement of Cl- and Gly ions. The two electric fields are in opposite directions and being different in magnitude, help the –vely charged proteins to stack at the interface of two gels. While entering resolving gel, as pH changes again, the Gly becomes –vely charge again and moves into the resolving gel quickly, effectively pushing the proteins into the resolving gel also. (@ Partha - In the absence of the stacking gel, this push will not be there. So all proteins will move into the gel at their own pace & give rise to a smear. But if stacking gel is present, all proteins in the sample get stacked & are pushed into the gel at the same time, to start resolving) Now in the resolving gel, the proteins separate based on molecular mass.
The function of the stacking gel is somewhat more complicated than many appreciate, and explained in the original Ornstein paper (doi:10.1111/j.1749-6632.1964.tb14207.x). In effect, an isotachophoresis (from Gr. 'migration at equal velocity') is performed, the stack (containing fast moving ions, proteins sorted by electrophoretic mobility and slow moving ion), once formed, moves at constant velocity. One can observe this beautifully with coloured marker proteins (e.g., "rainbow markers"), where the different proteins in the stack form slightly overlapping bands, like a roll of coins. Note that the proteins are ordered by mobility, not by size (order of colours is different in the stacking and resolving gel). The important thing is that the proteins get electrophoretically concentrated during stacking, and hence form very narrow bands, only a few µm thick (from several mm in the sample well). This causes the high resolution of discontinuous electrophoresis. I have described this process in detail in doi:10.1007%2F978-1-4419-7251-4_8
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