SDS-Polyacrylamide ElectrophoresisSDS聚丙烯酰胺凝胶电泳

1、.SDS-Polyacrylamide ElectrophoresisA. SDS-Polyacrylamide Electrophoresis Background. Proteins can be separated on the basis of size alone if they are first solubilized with the anionic detergent sodium dodecylsulfate (SDS). The SDS binds to the individual polypeptide chains comprising the protein, c

2、onverts the chains to rod-like shapes, and overwhelms the native protein charge with uniform SDS negatively charged groups. When subjected to polyacrylamide gel electrophoresis (PAGE) the polypeptide chains, which now have equal charge to mass ratios, are separated primarily according to molecular s

3、ize by the sieving effects of the gel. In SDS-PAGE, migration distance correlates semi-empirically with molecular weight, and a straight line should be obtained when plotting log MW vs. migration distance.For further information on SDS-PAGE, you may consult the original reference: Weber, K. and Osbo

4、rn, M. (1969), "The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis", J. Biol. Chem. 244: 4406-4412 (1969).B. SDS-PAGE Protocol.Solutions Used:Running Buffer: Purchase Pre-Made: Dilute to 1xProtein Sample Buffer: Purchase Pre-made 2xMole

5、cular weight standards: known molecular weights of standards used will be provided by your TAStaining Solution: 0.5% coomassie brilliant blue R-250 in 50% isopropanol, 50% acetic acidDestaining solution: 10% methanol, 10% acetic acidProcedure:Prepare samples for loading onto the gel 1. Dissolve 50 m

6、g/mL of protein in water (or buffer)2. Add 50mL of protein solution to 50mL of sample buffer3. Heat at 90ºC for 5 minutesAssemble the gel running unit1. Use pre-cast gel. Remove the comb gently, remove paper tab from bottom of pre-made gel. 2. Insert gel into running apparatus. Your TA will dem

7、onstrate how to do this.3. Add running buffer to the upper and lower portions of the assembly.Loading and running the gel1. Load 30 ml of each sample into the well. 2. Load 10 ml of known molecular weight standards in lane 1.3. Connect the power supply to the gel running assembly with correct polari

8、ty, and run your gel at a constant 150V.5. Your gel will run for 60 minutes.6. During this time your TA will give a lecture on SDS PAGE.Removing the gel and staining/destaining1. Once the run is complete, disconnect the power supply. You TA will demonstrate how to take the gel sandwich apart and transfer the gel into the staining container.2. Add Coomassie Blue R-250 solution and stain