What is SDS PAGE & how is it used to characterize proteins?

(Last Updated On: October 28, 2022)
Visualization of protein bands on SDS-PAGE gel.
Visualization of protein bands on an SDS-PAGE gel. Credit: piemmea via Common Wikimedia

What is SDS PAGE?

SDS PAGE or 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 an unknown protein using the SDS PAGE protocol.

SDS PAGE protocol requires five major components along with buffers and associated equipment. The major components are sodium dodecyl sulfate (SDS), acrylamide, bisacrylamide, ammonium persulfate, and TEMED. acrylamide and bisacrylamide are polymerized and cross-linked with each other to form a gel-like matrix. while APS and TEMED are the initiators of polymerization. TEMED catalyzes the breakdown of the APS into free radicals (sulfate free radicals) which helps in the polymerization and cross-linking of acrylamide and bisacrylamide into polyacrylamide. The last major component of is protocol is SDS which acts as a denaturant and provides charge to the protein sample

Characterization of proteins using SDS PAGE

The method of assaying SDS PAGE is called the SDS PAGE protocol. 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 the 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.

Components of the SDS-PAGE gel
Mix all these components in the test tube. Then add APS into the solution and dissolve it and then add TEMED immediately and shake well. After mixing, immediately load the gel into the glass plate gel caster and allow it to polymerization.

SDS PAGE protocol

Before starting the SDS PAGE protocol, 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 rod-shaped structures 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.

components of the gel buffer and running buffer
Formulation of the running buffer and gel buffers

Polyacrylamide gel formation

  • First, you need to prepare an 11 % resolving gel by mixing the required amount of components and then adding ammonium persulfate.
  • After adding the ammonium persulfate, immediately add the TEMED.
  • After pouring the resolving gel into the gel caster to 5cm, add a little amount of butanol at the top of the resolving gel to prevent the contact of oxygen with the gel (Oxygen prevents polymerization).
  • After polymerization of the resolving gel, remove the butanol with the help of tissue paper.
  • Then pour the freshly prepared 4 % stacking gel 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.
  • Now, load the protein marker on one side of the gel to make it differentiable and identifiable after staining and destaining.
  • After loading the protein samples into the respective well, run the gel by providing a constant voltage of 80-100 V.
polymerization of acrylamide and bisacrylamide
Mechanism of gel polymerization. Image: Ronald Mattern via Common Wikimedia

Electrophoresis, running the gel

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 the resolving gel. This difference is created by using different ionic strengths and the pH of the electrode buffer and stacking gel buffer. This phenomenon is called 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 at 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 with 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 toward 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 larger proteins. That’s because larger proteins get retarded successively by the 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 characterizing 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 the 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 do SDS PAGE protocol 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 of the protein, the sample may be subjected to Native PAGE. Subjecting native PAGE helps us determine 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 whether it has more than one polypeptide or not.

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