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Separating proteins using gel electrophoresis Electrophoresis is a relatively simple method to study the properties of proteins.

Separating proteins using gel electrophoresis Electrophoresis is a relatively simple method to study the properties of proteins.

Separating proteins using gel electrophoresis Electrophoresis is a relatively simple method to study the properties of proteins. It is based on the principle that charged molecules will migrate through a matrix when an electric field is applied.

The matrix used is polyacrylamide. NB Polyacrylamide is highly toxic, carcinogenic and teratogenic. The chemical agents used to form the polycarylamide matrix are acrylamide monomer and N, N methylene bisacrylamide. The size of the pores formed in the matrix depends on the amount of acrylamide per unit volume and the percentage of bisacrylamide. The normal range of polyacrylamide gels is 3-30%. Protein electrophoresis using polyacrylamide is sometimes called PAGE: polyacrylamide gel electrophoresis. There are two fundamentally different PAGE systems. These are dissociative (denaturing) and non-dissociative (non-denaturing). In non-dissociative PAGE, the proteins are maintained in their native comformations. Dissociative PAGE is designed to denature the proteins into their constituent polypeptides. This is usually done using a detergent called sodium dodecyl sulphate and hence the technique is called SDS-PAGE. This is the most common method for separating proteins based exclusively on size. In this class we will study dissociative (denaturing ) PAGE. Objectives: 1) understand the principles of electrophoresis 2) understand the use of protein standards for calculating protein mass 3) understand the difference between denaturing and non denaturing gel electrophoresis The demonstrator will explain each step of electrophoresis. You should watch carefully and make detailed notes in order to perform and write up the practical. You should describe each step of the process including any observations made while the gel is running. The protein sample will be the caco-2 extract that you produced in lab class 1. This must be completed Reagent Hazard Precautions Acrylamide Sodium dodceyl sulphate TEMED Ammonium persulphate Coomassie blue  Method 1) Cast the resolving gel (we did not prepare gel in this lab because we used ready and prepared gel) 4-12% BT ( NU-PAGE). 2) Pipette a volume of your sample that contains 5ug of protein into an eppendorf tube (you will need the concentration of the sample calculated in lab 1). Add 2.5ul of buffer and 2.5ul of water to make the total sample 10ul. 3) Boil the sample for 2 minutes and load it onto the gel along with protein standards provided 4) Resolve the gel at 150mv for 1 hour. 5) At the end of the time dismantle the gels and stain the gel in coomassie blue solution( instant blue) for 5 minutes. Destain and dry the gel . Address these specific points: 1) Describe the principle of electrophoretic separation of proteins (5marks) 2) what is the role of the following in SDS in SDS-PAGE: a) SDS b) B-mercaptoethanol c) TEMED and ammonium persulphate (5 marks) 3)Research the relationship between molecular weight and distance travelled on the gel. Using the model data supplied, calculate the weight of the unknown protein (20) 4)Describe the gel matrix and its effects on the migration of proteins. The recipe given is for a 12% gel. List the reagents and their volumes for a 10% gel. (5) 5) Describe how you would modify the procedure if you wanted to study native protein conformation (3) 6) describe the significance of the pH change between the two gels (2) 7) we used coomassie blue staining of proteins. Describe two other general protein stains. Under what conditions would they be used? (4 marks) 8) Research and describe a)2D electrophoresis. B) western blotting. In what situations might you use these?(12 marks) 9) Describe two clinical uses of protein electrophoresis. (10 marks) 10) The remainder of the mark is for the quality of the write up guidance as to how I want the reports presented. 1) Please DON’T use pen and ink as indicated in the handbook – send me the reports electronically. 2) You must complete the table of hazards for the chemicals used. 3) Report needs an introduction, a methods section (show all your calculations for making buffers), results , discussion and conclusions. U must use references for information and use Harvard style. 5) For the results, any graphs etc need to be made in a graph drawing programme such as Exel. You should plot means and standard deviation where appropriate. Please show all your working out for any calculations etc. 6) There are specific questions that need to be addressed – you can answer these in any appropriate section of the report.

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