Polyacrylamide Gel Electrophoresis: 

                  ⮚(Sodium dodecyl sulphate – polyacrylamide gel electrophoresis) SDS - PAGE is very commonly used separation technique for separating mixture of proteins based on their molecular weight or size.

               ⮚This system is based on gel base separation and the gel is complex of two type of gels – a resolving (running) gel in which proteins are resolved on the basis of molecular weight and a stocking gel in which the proteins are concentrated prior to the entering the resolving gel.

               ⮚This technique is widely used in different fields like biochemistry, forensic chemistry, biotechnology, research fields, genetics, and molecular biology. The gel here is stained with Coomassie staining or silver staining.

Gel electrophoresis Principle:

                Generally, the electrophoresis works on principle that charged particles move when electric field is applied. The proteins are made of long chain different amino acids and having a complex folded structure with high molecular weight. Proteins contain different charges based on the amino acids present, hydrophobic and hydrophilic nature. In SDS – PAGE the proteins are denatured to the primary structure and are negatively charged, so when the samples are loaded in the gel matrix the negatively charged molecules will move towards the anode (+).

Materials used in SDS - PAGE:

·        Buffer solution
·        Electrophoresis chamber and power supply
·        Comb
·        Casting frames
·        Protein samples
·        Buffer solutions
·        Polyacrylamide solution  
·        SDS

·        β-Mercaptoethanol

Procedure:

Sample Preparation: 

         ⮚The samples are mixed with β-Mercaptoethanol to breakdown the disulfide bond present in the poly peptide chain of the protein. After all the proteins attain the primary structure. β-Mercaptoethanol is a reducing agent which reduces the S-S bond responsible for secondary structure.

        ⮚While preparing sample for SDS – PAGE the sample buffer is mixed with SDS (sodium dodecyl sulphate). SDS imparts the negative charge to all the protein molecules in the sample. So, when electric field is applied all the negatively charged proteins moves towards the anode (+).

       ⮚Bromophenol blue (BPB) is mixed with the sample proteins. It works as a tracking dye in the electrolysis process and it is used to monitor the progress of the samples during the electric field is applied. BPB is negative charge so it travels along with the sample. After the addition of SDS and β-Mercaptoethanol the sample is boiled for sometimes.

Preparation of Polyacrylamide Gel:

       ⮚The polyacrylamide gel is prepared by the polymerization of acrylamide in the distilled water with small amount of cross linker like N,N-Methylene bisacrylamide.

       ⮚The two layers have different functions: the stacking gel is needed to concentrate/pack all the proteins in one line, so that they will start migrating in the running gel all at the same time. The running gel allows to separate the proteins in the sample based on their molecular weight. 

Resolving Gel:

       ⮚It has a PH 0f 8.8, Resolving gels has a smaller pore allows to migrate the protein based on their molecular weight and having higher ionic strength. For the preparation of 14% running gel mix the components in the following order - Distilled water: 10.33 ml, tris buffer PH (8.8): 10 ml, Acrylamide/bisacrylamide 30%: 18.67 ml, 10%SDS: 400 µl, 10% APS ( Ammonium persulphate ): 200 µl, TEMED ( tetramethylethylenediamine ): 40 µl, Total = 40 ml.

       ⮚After mixing the components immediately pour the mix in between the plates, fill the space up to there will be enough room to form stocking gel of 0.5 to 1cm. Overlay with 70% ethanol and allow the gel to polymerize for sometimes after that rinse the ethanol from the surface with distilled water.

Stacking Gel:

         It has a PH of 6.8, generally the stocking gel has larger pores allows the proteins to migrate freely and lower ionic strength. For the preparation of 4% of stacking gel mix the components in the following order – Distilled water: 3.35 ml, tris buffer PH (6.8): 2.5ml, Acrylamide/bisacrylamide 30%: 4.0 ml, 10%SDS: 100 µl, 10% APS: 50 µl, TEMED: 15 µl, Total = 10 ml. After mixing the components immediately pour the mix in between the plates and insert the comb in it. Allow the gel for polymerization up to 30 mins after that remove the comb and place it in the electrophoresis chamber. Pour the running buffer in the bottom and top of the chamber.

Electrophoresis:

         ⮚The gel matrix is placed in the electrophoresis chamber, and the chamber is filled with buffer solutions. While running an SDS-PAGE gel we use 3 buffers, Tris- Gly (8.3), Tris-Cl (pH 6.8) & Tris-Cl (8.8). The Tris-Cl buffers are present in the stacking & resolving gels respectively. The Tris-Gly is the buffer used for running the apparatus.

         ⮚In Tris-Gly at pH 8.3 the glycine exists as a –ve charge and moves towards the positive electrode. So, this –ve charged Gly enters the stacking gel. In the stacking gel, the pH changes to 6.8 where Gly exists in zwitter-ionic form.

         ⮚The electrodes are connected at each end and the protein samples are loaded in the wells of the vertically placed gel. After loading the samples in the wells, the electric field is applied in a uniform range. The negatively charged particles starts to move towards anode (+) through the pores of the gel. The tracking dye shows the progress of the electrophoresis once the band reached the bottom the electric field is cut offed. The gel matrix is now taken out for the staining process or may enter in the western blotting.

Applications: 

·        Estimation of protein purity
·        Used in peptide mapping
·        Measures the molecular weight of the molecules
·        It is used in western blotting

·        Used in the HIV tests to separate the HIV proteins and lot of applications are there.

References