Learn SDS PAGE Practically, Staining, Results, Everything in one video. скачать в хорошем качестве

Learn SDS PAGE Practically, Staining, Results, Everything in one video.

SDS PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for the separation of proteins based on their molecular weight. It is a technique widely used in forensics, genetics, biotechnology and molecular biology to separate the protein molecules based on their electrophoretic mobility. Principle of SDS-PAGE The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the opposite sign when placed in an electric field. The separation of the charged molecules depends upon the relative mobility of charged species. The smaller molecules migrate faster due to less resistance during electrophoresis. The structure and the charge of the proteins also influence the rate of migration. Sodium dodecyl sulphate and polyacrylamide eliminate the influence of structure and charge of the proteins, and the proteins are separated based on the length of the polypeptide chain. Role of SDS in SDS-PAGE SDS is a detergent present in the SDS-PAGE sample buffer. SDS along with some reducing agents function to break the disulphide bonds of proteins disrupting the tertiary structure of proteins. Materials Required: Power Supplies: It is used to convert the AC current to DC current. Gels: These are either prepared in the laboratory or precast gels are purchased from the market. Electrophoresis Chambers: The chambers that can fit the SDS-PAGE gels should be used. Protein Samples: The protein is diluted using SDS-PAGE sample buffer and boiled for 10 minutes. A reducing agent such as dithiothreitol or 2-mercaptoethanol is also added to reduce the disulfide linkages to prevent any tertiary protein folding. Running Buffer: The protein samples loaded on the gel are run in SDS-PAGE running buffer. Staining and Destaining Buffer: The gel is stained with Coomassie Stain Solution. The gel is then destained with the destaining solution. Protein bands are then visible under naked eyes. Protein Ladder: A reference protein ladder is used to determine the location of the protein of interest, based on the molecular size. Protocol of SDS-PAGE Preparation of the Gel All the reagents are combined, except TEMED, for the preparation of gel. When the gel is ready to be poured, add TEMED. The separating gel is poured in the casting chamber. Add butanol before polymerization to remove the unwanted air bubbles present. The comb is inserted in the spaces between the glass plate. The polymerized gel is known as the “gel cassette”. Also read: Applications of Biotechnology Sample Preparation Boil some water in a beaker. Add 2-mercaptoethanol to the sample buffer. Place the buffer solution in microcentrifuge tubes and add protein sample to it. Take MW markers in separate tubes. Boil the samples for less than 5 minutes to completely denature the proteins. Electrophoresis The gel cassette is removed from the casting stand and placed in the electrode assembly. The electrode assembly is fixed in the clamp stand. 1x electrophoresis buffer is poured in the opening of the casting frame to fill the wells of the gel. Pipette 30ml of the denatured sample in the well. The tank is then covered with a lid and the unit is connected to a power supply. The sample is allowed to run at 30mA for about 1 hour. The bands are then seen under UV light. Applications of SDS-PAGE The applications of SDS-PAGE are as follows: It is used to measure the molecular weight of the molecules. It is used to estimate the size of the protein. Used in peptide mapping It is used to compare the polypeptide composition of different structures. It is used to estimate the purity of the proteins. It is used in Western Blotting and protein ubiquitination. It is used in HIV test to separate the HIV proteins. Analyzing the size and number of polypeptide subunits. To analyze post-translational modifications.