More importantly, these charges swamp the inherent charge of the proteins and give every protein the same charge-to-mass ratio. Because the proteins have the same charge-to-mass ratio, and because the gels have sieving properties, mobility becomes a function of molecular weight. But what about running gels, stacking gels, electrode buffer, and all these different pHs?
The velocity of a charged particle moving in an electric field is directly proportional to the field strength and the charge on the molecule and is inversely proportional to the size of the molecule and the viscosity of the medium. Adding a gel with sieving properties that is a gel where the resistance to the motion of a particle increases with particle size increases the differences in mobility between proteins of different molecular weights. This is the basis of separation.
The problem now becomes how to line up all the proteins in an orderly fashion at the starting gate. Laemmli gels are composed of two different gels stacker and running gel , each cast at a different pH. In addition, the gel buffer is at a third, different pH. The running gel is buffered with Tris by adjusting it to pH 8. The stacking gel is also buffered with Tris but adjusted to pH 6.
The sample buffer is also buffered to pH 6. The electrode buffer is also Tris, but here the pH is adjusted to a few tenths of a unit below the running gel in this case 8. We run our gels at constant voltage. To remove charge as a factor in protein migration through the gel. SDS binds to proteins with high affinity and in high concentrations.
This results in all proteins regardless of size having a similar net negative charge and a similar charge-to-mass ratio. In this way, when they start moving through a gel, the speed that they move will be dependent on their size, and not their charge. It is by far the biggest factor. However, SDS can bind differently to different proteins. Hydrophobic proteins may bind more SDS, and proteins with post-translational modifications such as phosphorylation and glycosylation may bind less SDS. These effects are usually negligible, but not always, and should be considered if your protein is running at a different molecular weight than expected.
What is in the running buffer? Tris, glycine, and SDS, pH 8. Its pKa of 8. This makes it a good choice for most biological systems. SDS in the buffer helps keep the proteins linear. Glycine is an amino acid whose charge state plays a big role in the stacking gel.
More on that in a bit. What is in the sample loading buffer? This is the buffer you mix with your protein samples prior to loading the gel.
Again with the Tris buffer and its pKa. The SDS denatures and linearizes the proteins, coating them in negative charge. 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.
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. Proteins migrate at different rate depending on the concentration of the separating gel. Use an appropriate gel concentration for your target protein.
Using a higher acrylamide concentration produces a gel with a smaller mesh size suitable for the separation of small proteins. 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 Has this helped you?
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