How to Perform SDS-PAGE to Characterize Different Proteins

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(Last Updated On: April 23, 2017)
Visualization of protein bands on SDS-PAGE gel.

Visualization of protein bands on SDS-PAGE gel. Credit: piemmea via Common Wikimedia


SDS-PAGE, Sodium Dodecyl Sulfate Polyacryl Amide Gel Electrophoresis is an analytical tool widely used to analyze protein mixtures qualitatively as well as quantitatively. It can also be used to determine the molecular weight of the protein.

This method is based on the separation of proteins according to their molecular size. It can be used to demonstrate the presence of a single type of polypeptides whether there are subunits or not or different polypeptides. The relative molecular weight of the protein run through SDS-PAGE can be calculated based on the calibration curve drawn using molecular weight of standard marker proteins and RF (retention factor of the marker proteins). SDS-PAGE is a denaturing electrophoresis where proteins are denatured to their primary structure (polypeptide chain) and all other structures are lost.

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, immidiately load the gel into the glass plate gel caster and allow it for polymerization.

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 will be fully denatured and will become 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.

components of the gel buffer and running buffer

Formulation of the running buffer and gel buffers

Polyacryl Amide Gel Formation

At first, the resolving gel is prepared using 11% by mixing required amount of components then adding APS (ammonium persulfate) and at the last (before pouring the gel into the caster) TEMED is added. The resolving gel is poured into the glass place gel caster up to 5cm and butanol is added at the top of the resolving gel to prevent the contact of oxygen with the gel which prevents the polymerization.

After a few minutes when resolving gel is polymerized, butanol is removed completely using tissue paper. The stacking gel is prepared with 4% concentration and similarly loaded at the top of the resolving gel up to 0.8 cm height. Then, a comb is placed to create wells in the gel. After solidification gel is placed in the tank, the running electrode buffer is added into the tank and comb from the gel is removed.

The protein sample is loaded into the wells of the gel, while the standard protein marker is loaded at one side of the gel to make it differentiable and identifiable after staining and destaining.

polymerization of acrylamide and bisacrylamide

Mechanism of gel polymerization. Image: Ronald Mattern via Common Wikimedia

Electrophoresis, Running the Gel

After loading the sample proteins, the gel is run by providing constant voltage 80-100 V. The purpose of stacking gel is to stack the protein samples (to concentrate) into a sharp band before interning the main separating gel. However, the pore size of the stacking gel is larger as compared to that of resolving gel (separating gel) that is created by using different in ionic strength and the pH of electrode buffer and stacking gel buffer. This phenomenon is called as isotachophoresis.

The stacking gel has a large pore size because it is made of 4 % acrylamide and, therefore, it allows the protein to move freely and concentrate under the influence of electric field. The band sharpening takes place by the negatively charged glycinate ions present in 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.

When current supply is turned on 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 glycinate ions reach the separating gel it becomes fully ionized in the higher pH environment and its mobility increases. The pH of the stacking gel is 6.8 while of the separating gel is 8.8. Thus, 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. As 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 and proteins are separated.

Small proteins move faster than that of smaller proteins because larger proteins get retarded successively by frictional resistance due to the sheaving effect of the gel. As bromophenol blue (which has been used as a tracking dye) is small it is not retarded and reaches the bottom of the gel. The current is turned off and the gel is removed.

Now it’s time to remove the gel from the glass plate and stain the gel using the staining solution for an appropriate time and then washed with a destaining solution for an appropriate time to remove the dye bound to the gel to visualize the protein bands.

Optimization of SDS-PAGE to Characterize Proteins of Different Molecular Weight

Generally, 15% polyacrylamide gel is used as separating gel and the pore size formed will allow the separation of proteins of molecular mass 10,000 unhindered. Therefore, 15% gel is used to separate proteins in the range of Mr 100000 to 10,000. However, separation of proteins of molecular weight 200,000 to 15,000, 10 % polyacrylamide gel is used.

The molecular weight of a protein can be determined by comparing its mobility with those of the standard marker proteins of known molecular weight run together with the same gel. A graph of distance moved by standard proteins against the log of their molecular weight is plotted. A calibration is obtained with the help of the equation of that curve, the molecular weight of the sample is calculated.

SDS-PAGE gel electrophoresis is often used to study and characterize the nature of proteins after each step of purification to access the purity of the sample. Therefore, a pure protein should give single band on SDS-PAGE until and unless the protein is of multimeric of different unequal subunits.

If there are more than one band obtained in SDS-PAGE after purification the protein sample may be subjected to Native-PAGE. This determines whether bands obtained in SDS-PAGE are of different subunits or of different polypeptides. As Native-PAGE gives a single band for a single protein, no matter it has more than one polypeptide or not.

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