SDS PAGE II Protein Electrophoresis- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

This video contains 1- SDS PAGE sample preparation 2-Significance of SDS/ Role of SDS 3-Significance of 2-mercaptoethanol/ role of 2-mercaptoethanol 4- Significance of Glycerol/ Role of Glycerol 5-Significance of tracking dye/ Role of Tracking dye 6-Significance of TEMED/ Role of TEMED 7-Significance of APS/ Role of APS 8-Significance of acrylamide/ Role of acrylamide 9-Significance of bis-acrylamide/ Role of bis-acrylamide 10- SDS PAGE gel preparation 11- Polymerization of SDS PAGE gel 12-Principle of SDS PAGE 13- Detection of proteins using coomassie brilliant blue/ silver staining 14- Application of SDS PAGE SDS–polyacrylamide gel electrophoresis (SDS–PAGE) is the most widely used method for analysing protein mixtures qualitatively. It is particularly useful for monitoring protein purification Samples to be run on SDS–PAGE are firstly boiled for 5 min in sample buffer containing b-mercaptoethanol and SDS. The mercaptoethanol reduces any disulphide bridges present that are holding together the protein tertiary structure, and the SDS binds strongly to, and denatures, the protein. Each protein in the mixture is therefore fully denatured by this treatment and opens up into a rod-shaped structure with a series of negatively charged SDS molecules along the polypeptide chain. On average, one SDS molecule binds for every two amino acid residues. The original native charge on the molecule is therefore completely swamped by the negatively charged SDS molecules The sample buffer also contains an ionisable tracking dye, usually bromophenol blue, that allows the electrophoretic run to be monitored, and sucrose or glycerol, which gives the sample solution density thus allowing the sample to settle easily through the electrophoresis buffer to the bottom when injected into the loading well Once the samples are all loaded, a current is passed through the gel and the proteins are separated. Video transcript: SDS-PAGE is mainly carried out for the separation of proteins. For carrying out SDS-PAGE, the sample, i.e. the protein samples are prepared using tris-buffer which maintains the pH, glycerol which gives the sample enough density so it settles inside the well. Bromophenol blue which is a tracking dye, 2-mercaptoethanol and SDS. A protein molecule exists in a folded secondary or tertiary structure in its native form. In these structure disulfide bonds are formed between the sulfur residues on two cysteine molecules. b-mercaptoethanol basically reduces these di-sulfide bonds, leading to a denatured unfolded form of protein. this, denaturation is triggered by heat. SDS binds to the unfolded protein and solubilizes it. SDS also provides an overall negative charge to the entire protein. Approximately 1 SDS molecule binds to 2 amino acids. Proteins differ from each other on the basis of size as well as charge. But since the proteins are denatured with an overall negative charge due to SDS. the movement on the gel will not be based on the charge of the protein, but only on the size of the protein. the concentration of the gel depends on the amount of acrylamide added in the buffer. Thus, the pore size of the gel, depends upon the concentration of acrylamide that is used to prepare the gel. Saturated butanol, or isopropanol is added to avoid oxygen and to facilitate proper polymerization of the gel matrix. A polyacrylamide gel is made up of acrylamide and bis-acrylamide monomers which are polymerized to form the gel matrix. the polymerization is done using TEMED and APS. TEMED decomposes Ammonium persulfate to give free radicals. Free radicals species are highly reactive, they have an unpaired electron, and thus they form single bond with another monomers. this creates an equally reactive radical which will react with another monomer and form long chains. The long chains are cross-linked by bis-acrylamide. The stacking gel has low acrylamide concentration, i.e. it has a high pore size that allows the proteins to migrate freely and get stacked between the interface of the stacking gel and the running gel. The resolving gel contains a high concentration of acrylamide, i.e. it is having less pore size, which is capable of retarding the movement of proteins. Thus, it is the resolving gel where the separation of protein molecules takes place. Another difference between the stacking gel and the resolving gel lies in the pH. Stacking gel pH= 6.8 Resolving gel pH= 8.8 Running buffer pH= 8.3 Glycine can exist in three different charged staged, i.e. positive, neutral, or negative depending on the pH. Low pH= positive charge High pH= negative charge Neutral pH- zwitter ion The proteins move in this sandwich manner till it hits the running gel. After the gel is run, it can be removed and stained using coomasie brilliant blue, or silver nitrate to visualize the protein. Western blot can also be carried out with the gel.