Methods of Gel Electrophoresis Flashcards
How is agarose gel prepared?
Add agarose to the required percentage in TAE buffer. Dissolve agarose by boiling and pour into a casting tray to let it cool down and solidify.
What is the composition of the loading buffer used in SDS-Polyacrylamide Gel Electrophoresis of Proteins?
Loading buffer contains glycerol and tracking dye: bromophenol blue. Also contains SDS and DTT or β-mercaptoethanol.
How are samples treated before loading in Non-reducing Polyacrylamide Gel Electrophoresis of Proteins?
Samples boiled for a few minutes in loading buffer.
Which electrode do molecules move towards in Native Polyacrylamide Gel Electrophoresis of Proteins?
Positive.
What is the principle of biomolecule separation in Agarose Gel Electrophoresis of DNA?
Agarose polymer forms a meshwork allowing DNA separation by size.
Which stain is commonly used to observe bands in SDS-Polyacrylamide Gel Electrophoresis of Proteins?
Coomassie blue.
Describe the process of gel preparation in SDS-Polyacrylamide Gel Electrophoresis of Proteins.
There are usually two layers, the stacking gel and the resolving gel. Both gels contain Acrylamide and bis-acrylamide. To cause “gelling,” one uses TEMED to catalyze the formation of persulfate radicals from ammonium persulfate. The radicals will cause acrylamide and bisacrylamide to cross-link leading to solidification of the gel.
What is the treatment of samples prior to loading in Native Polyacrylamide Gel Electrophoresis of Proteins?
Samples boiled for a few minutes in loading buffer.
How are proteins separated in Non-reducing Polyacrylamide Gel Electrophoresis of Proteins?
Protein is denatured and coated with negatively charged SDS which masks their intrinsic charges. Mobility is a linear function of the logarithms of their molecular mass.
Which tracking dye is commonly found in loading buffer used in Agarose Gel Electrophoresis of DNA?
Bromophenol blue.
