Because of the hydrophobic effect, the surfaces of proteins proteins have a higher frequency of polar and charged amino acids than the interiors, where hydrophobic residues predominate. Folded proteins assume many different geometries and their surfaces are mosaics with respect to the distribution of R groups with different chemistries.
Because proteins are so diverse with respect to their surface charges and geometries, the molecular weights of folded proteins cannot be simply determined by their migration rate in an electric field. Postively and negatively charged proteins would migrate in different directions! The combination of heat and detergent is sufficient to break the many noncovalent bonds that stabilize protein folds, and 2-mercaptoethanol breaks any covalent bonds between cysteine residues.
Like other detergents, SDS is an amphipathic molecule, consisting of a hydrophobic carbon chain and a hydrophilic sulfate group. The SDS hydrocarbon chain permeates the protein interior and binds to hydrophobic groups, reducing the protein to a random coil, coated with negatively charged detergent molecules all along its length. The stacking and running resolving gels have different pore sizes, ionic strengths and pHs.
The third component is the electrophoresis buffer 25 mM Tris, mM glycine,, 0. The ionization state of the glycine is critical to the separation. At neutral pH, glycine is a zwitterion, with a negatively charged carboxyl group and a positively charged amino group. The pK a of the amino group is 9. Consequently, very little glycine has a negative charge in the chamber buffer or stacking gel, and significant ionization does not occur until the glycine enters the more alkaline pH 8.
Th e sample buffer also contains glycerol, which allows the protein samples to settle into the bottom of the gel wells. The gel is vertically positioned in the electrophoresis apparatus and covered with chamber buffer containing glycine right, shaded. Once a voltage is applied, the chloride ions in the sample buffer and stacking gel move rapidly toward the positive pole, forming the leading edge of a moving ion front. Glycine molecules have very little charge in the stacking gel, so they migrate at the rear of the moving ion front.
This difference in chloride and glycine mobility sets up a steep voltage gradient in the stacking gel that sweeps along the negatively charged protein-SDS complexes. Protein-SDS complexes remain concentrated at the interface until the slowly migrating glycine molecules reach the boundary between the two gels. Dramatic changes occur as the glycine ions enter the running gel. The pH of the running gel is closer to the pKa of the glycine amino groups, so a significant fraction of the glycine molecules assume a negative charge.
Negatively charged glycine molecules begin to move at the same rate as the chloride ions, thereby eliminating the voltage difference that controlled protein mobility through the stacking gel. The pores in the running gel are much smaller than those of the stacking gel, so the pores present frictional resistance to the migration of proteins. Proteins begin to migrate at different rates, because of the sieving properties of the gel. Within a certain range determined by the porosity of the gel, the migration rate of a protein in the running gel is inversely proportional to the logarithm of its MW.
This is because there are two hydrogen atoms for every one oxygen in a water molecule. There will be twice as many hydrogen gas molecules formed. Application of SDS to proteins causes them to lose their higher order structures and become linear.
What exactly does SDS do? It unfolds proteins. Since SDS is anionic negatively charged , it binds to all the positive charges on a protein, effectively coating the protein in negative charge. Why do we want the protein coated in negative charges?
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. 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. Typically, the system is set up with a stacking gel at pH 6. Figure 1. Well, glycine can exist in three different charge states: positive, neutral, or negative, depending on the pH.
This is shown in the diagram below. Control of the charge state of the glycine by the different buffers is the key to the whole stacking gel thing. When the power is turned on, the negatively charged glycine ions in the pH 8.
In this environment, glycine switches predominantly to the zwitterionic neutrally charged state. This loss of charge causes the glycine ions to move very slowly in the electric field. 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.
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