Polyacrylamide Gel Electrophoresis (Sds-page Method)

POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE METHOD) Protein is a polymer whose monomers consist of acids Aminos are linked via peptide bonds. An active protein is a polypeptides that have folded to form tertiary and quaternary structures. Proteins Certain substances have a certain composition and amount of nimo acid. In other words Proteins have a certain molecular weight and are usually expressed in units Da (Dalton). One amino acid molecule has a molecular weight of 110 Da. Heavy A protein molecule can be determined in various ways, one of which is by electrophoresissodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The SDS-PAGE method can also be used to determine whether A protein consists of one or more subunits. The SDS-PAGE method is a protein separation based on differences the molecular weight of a protein in an electric field. SDS is a detergent that negatively charged which will form a complex with protein molecules. Thus the protein will be negatively charged with a comparable charge with the length of the subunit chain. SDS binds to hydrophobic parts of proteins and damage the secondary, tertiary and quaternary structures of proteins. Polyacrylamide gel is supporting medium used to separate proteins. This gel is the result polymerization of acrylamide using N,N'methylene-bisacrylamide as cross-link forming. Protein molecules will migrate through the gel pores acrylamide with mobility that depends on its length. If a protein contains more than one subunit then electrophoresis there will be more than two molecules migrating. The molecular weight of the protein can bendetermined using a calibration curve between logarithms of molecular weight standard proteins and migration distance. This SDS-PAGE electrophoresis technique can also be used for various purposes, for example: determining protein purity, determining protein concentration, determination of proteolysis and detection of modifications to proteins. This SDS-PAGE electrophoresis technique can also be used for various purposes, for example: determining protein purity, determining protein concentration, determination of proteolysis and detection of modifications to proteins. 1. Experiment Tool : 1. Bio-Rad Mini-Protean II 2. Power Supply 3. Eppendorf tube 4. Gel loading tips Material : 1. Acrylamide: bis-acrylamide (39 :1) 2. Tris-HCl pH 6.8 lM 3. Tris-HCl pH 8.7 lM 4. SDS 20% 5. N, N, N”,N”-tetramethylethylenediamine (TEMED) 6. Ammonium persulfate 10% 7.Running buffer(1 gr SDS 3.03 gr Tris, 14.4 gr glycine, add water up to 100 ml) 8. Solutionstanning940 ml methanol, 15 ml acetic acid, 0.1 gr Coomasie blue, add water up to 100 ml) 9. Solutiondestaining(10 ml methanol, 7.5 ml acetic acid, water added up to 100 ml) 10. 5 xSample buffers(12.5 ml 1 M tris pH 6.8 20 ml, glycerol, 10 ml mercaptoethanol, 40 ml 10 % SDS, 15 mgBromophenol Blue. Procedure: A. Gel Preparation 1. Mix all the solutions forseparating gel in order of addition according to Table 1. 2. Pour (using a pipette) the solution intosandwich gel until approximately 1.5 cm from the top of the plate. 3. Add water until the height is approximately 1 – 5 mm. 4. Leave until polymerization occurs (30 minutes) 5. Mix all the solutions forstacking gel in order of addition according to the table above. 6.Discard the water at the topseparating gel. 7.Pour the mixturestacking gel onseparating gel and insertcomb 8. Leave until polymerization occurs (30 minutes) B. Sample Preparation 1. Prepare standard protein solutions and protein samples 2. Add 5 x sample buffer solution 3. Boil for 5 minutes 4. Cool in ice for 5 minutes C. Gel Electrophoresis 1. Pairsandwich gel which has been prepared using an electrophoresis apparatus. 2. Pourrunning bufferinto thetank electrophresis 3. Insert the protein sample (10-20 µg) into each gel hole with a pipette micro( Eppendorf ) 4. Install the electrophoresis device cover. 5. Connect to the power supply and perform electrophoresis at a fixed voltage 200v 6. Turn off the device afterblue dyeright out of the gel (approx. 60 minutes) D. Staining and Destaining 1. Remove the gel fromplates(use gloves) 2. Put it in the solutionstaining ( 20ml) 3. Leave it for 15 minutes 4. Transfer into the solutiondestaining ( 50 ml) 5. Leave it until the gel becomes clean (30 minutes- overnight) Task : 1. Create a standard protein calibration curve. 2. Determine the molecular weight of the protein of the sample being analyzed. Question : Explain the reaction for the formation of polyacrylamide polymers and state the function of biacrylamide. TEMED, ammonium persulfate and mercaptoethanol. Explain the principle of electrophoresis Explain how to visualize proteins separated using SDSPAGE