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SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis is an analytical tool widely used to analyze protein mixtures qualitatively as well as quantitatively. However, we can also use it to determine the molecular weight of the protein.
The given method is based on the separation of proteins according to their molecular size. We can also use it to demonstrate the oligomeric nature of the polypeptide. It means that SDS-PAGE can help us to know whether the protein is multimeric of the same polypeptides or of different polypeptides. The relative MW of the protein run through SDS-PAGE can be calculated based on the calibration curve. The calibration curve is drawn using MW of standard protein markers and the retention factor of the marker proteins. SDS-PAGE is denaturing electrophoresis where SDS and beta-mercaptoethanol denature the protein structures to their primary structure.
Sodium Dodecyl Sulfate, an anionic detergent
SDS is an anionic detergent. The sample to be run needs to be boiled for five minutes in sample buffer(prepared by mixing 3.55 ml deionized water, 1.25 ml of stacking gel buffer, 2.5 ml glycerol, 2 ml 10 % (w/v) SDS, 0.2 ml 0.5 % (w/v) bromophenol blue and 50μl β-mercaptoethanol). The sample buffer with heating is used to ensure that proteins are denatured and SDS molecules get bound to the protein chain. β-mercapto-ethanol just cleaves the disulfide bond present.
Therefore, each protein present in the sample gets fully denatured. Protein molecules become a rod-shaped structure with an equal charge to mass ratio. This charge will be provided by the SDS. It is assumed that one molecule of SDS binds to every two amino acid residues. This negative charge present in the straight line structure of polypeptide prevents the protein to fold back into the 3d structure.
The sample buffer is also mixed with tracking dye bromophenol blue and glycerol. Glycerol provides density to the protein sample, thus allowing the sample to settle down in the well of the gel. While bromophenol blue provides color, that can be tracked into the gel to determine the movement of the sample through the gel.
Polyacrylamide gel formation
At first, prepare the 11 % resolving gel by mixing the required amount of components and then add ammonium persulfate. After adding the ammonium persulfate, immediately add the TEMED. Pour the resolving gel into the gel caster to 5cm. After that, add a little amount of butanol at the top of the resolving gel. It prevents the contact of oxygen with the gel which prevents the polymerization.
After polymerization of the resolving gel, remove the butanol with the help of tissue paper. Then pour the freshly prepared 4 % stacking gel load at the top of the resolving gel up to 0.8 cm height. Then, place a comb to create the desired number of wells in the gel. Once the stacking gel solidifies, place the gel into the tank. Put the running electrode buffer into the tank.
Remove the comb from the gel and load the protein samples into the wells of the gel. Load the protein marker on one side of the gel to make it differentiable and identifiable after staining and destaining.
Electrophoresis, running the gel
After loading the protein samples, run the gel by providing a constant voltage of 80-100 V. The purpose of stacking gel is to concentrate the protein samples into a sharp band before interning the main separating gel. However, the pore size of the stacking gel is larger than that of resolving gel. This difference is created by using different ionic strength and the pH of the electrode buffer and stacking gel buffer. This phenomenon is called as isotachophoresis.
The stacking gel has a large pore size because of less concentration (4 % ) of acrylamide. Therefore, it allows the protein to move freely and concentrate under the influence of the electric field. The band sharpening takes place by the negatively charged glycinate ions present in an electrode buffer. As glycinate ion has low electrophoretic mobility than protein-SDS complex while Cl- ion present in loading buffer and the stacking gel buffer has high mobility.
Sandwich-like structure of glycinate-protein-Cl complex
When we turn on the current supply, all the ionic species start to migrate with the same speed otherwise, there would be a break in the electrical circuit. As field strength is inversely proportional to conductivity which is proportional to the concentration of the ions. Thus, these three species of interest will form a sandwich-like structure lower chloride ions, upper glycinate ions and in between them protein-SDS complex.
As soon as glycinate ions reach the separating gel, it becomes fully ionized in the higher pH. Its mobility increases in the higher pH environment. The pH of the stacking gel is 6.8 while the separating gel is 8.8. Therefore, a protein-SDS complex is left behind the Cl– ion and glycinate ion. Now, this protein-SDS complex moves on its own and continues to move towards the anode. Since protein-SDS complexes have the same charge per unit length, they travel into the separating gel with the same mobility. However, they pass through the separating gel where proteins are separated.
Small proteins move faster than that of larger proteins. That’s because larger proteins get retarded successively by frictional resistance/sheaving effect of the gel. The tracking dye; bromophenol blue is a small molecule and will not retard and reaches the bottom of the gel. Once the tracking dye reaches the limit, turn off the current and remove the gel for staining and destaining processes.
Now it’s time to remove the gel from the glass plate and stain the gel using the staining solution. After staining for 2-3 hours, wash the gel with a destaining solution for 3-4 hours or overnight. During the destaining process, proteins in the well will appear as blue bands representing protein subunits.
Optimization of SDS-PAGE for the characterize proteins of different molecular weight
Generally, separating gel contains 15 % polyacrylamide. The pore sizes of 15 % polyacrylamide gel can allow the separation of proteins of MW 100,000 to 10,000 Da unhindered. In the same way, we can use 10 % polyacrylamide gel to separate proteins of MW from 200,000 to 15,000 Da.
We can easily estimate the MW of a protein by comparing its mobility with those of the standard protein markers. To do so, we need to run the protein markers along with the sample in the same gel. After the run, make a graph of distance moved by standard proteins against the log of their molecular weight. The calibration graph gives a linear regression equation that we can use to calculate the molecular weight of the protein sample.
We often SDS-PAGE to study and characterize the nature of the protein after each step of purification. It helps us to access the purity of the sample. Therefore, a pure protein should give a single band on SDS-PAGE. If you see more than one band, it may be due to the fact that protein is oligomeric of unequal subunits.
If there is more than one band in SDS-PAGE after purification the protein sample may be subjected to Native-PAGE. Subjecting native-PAGE helps us determines whether bands obtained in SDS-PAGE are of different subunits or of different polypeptides. Native-PAGE gives a single band for a single protein, no matter it has more than one polypeptide or not.